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Special Issue

Integrating Phylogenies into Community Ecology1

The organisms that live together in a community do so both because they are present in the larger regional pool and because they have characteristics that permit their existence at that locality and their coexistence with other species in the community. Neither a species’ regional presence nor its characters can be fully understood without taking the species’ history into account. That history is contingent on chance events and on deterministic interactions with other species in historical communities. As this historical approach gains favor in ecology, and as our understanding of the tree of life expands, ecologists and systematists are increasingly working together. However, this new partnership often requires synthesizing ideas across disciplines. Our goal with this Special Issue is to explore the practical interchange of concepts between evolutionary biology and community ecology, highlighting studies that both use phylogenetic information and consider the community context of individual organisms, and that represent a range of disciplines, from microbiology and parasitology to ornithology. Several common threads weave through the papers. The first concerns the importance and definition of the local community itself. As one steps back and takes a historical and biogeographic view of a species, averaging over variation in local community composition across its range, local interspecific interactions appear less influential for the species’ evolution. Ricklefs, in considering the causes of variation in the emergent property of community species richness, goes so far as to say that ‘‘ecologists [must] abandon the idea of the local community.’’ At the same time, however, there is abundant evidence that inter-individual interactions do influence which particular taxa co-occur (e.g., Webb et al.), alter their ranges, and under certain circumstances, lead to evolutionary adaptation that reduces negative interspecific interaction. Separating the effects of local processes on regional patterns and regional processes on local patterns will always be hard. However, with large and numerous samples we can come to understand variation in community structure over wide areas. Lovette and Hochachka draw on the vast Breeding Bird Survey data set to examine both local and regional composition in warbler communities, and Kembel and Hubbell examine phylogenetic community structure of trees at varying scales within the 50-ha BCI Forest Dynamics Plot. As well as drawing spatial boundaries around communities, we are forced to define their taxonomic bounds. Cavender-Bares et al. demonstrate how increasing the ‘‘phylogenetic scale’’ of communities influences our understanding of their phylogenetic structure. Brooks et al. and Weiblen et al. address the complex question of the phylogenetic structure of compound communities, with platyhelminth parasites of anurans, and insect herbivores of plants, respectively. The second key thread dealt with by many authors is, ‘‘what exactly are ecological characters, and how do they evolve?’’ For ecologists, it is obvious to ask how an organism’s niche has evolved and to treat it as a character to be reconstructed on a phylogeny. However, systematists often argue that because the habitats and realized niches that we can observe are influenced by interspecific interactions and community composition, they are not actually evolvable entities. Instead we should decompose overall niches into directly heritable, morphological, and physiological characters. For example, Agrawal and Fishbein show how defensive syndromes involve combinations of numerous characters of plants, and Fine et al. show that defensive traits evolve in a trade-off with growth. Different components of the overall niche may also be subject to different ecological interactions: an organism might occupy a habitat that conforms to a niche on one environmental axis, while competition within a habitat might lead to resource partitioning on another axis. Silvertown et al.

1 Reprints of this 166-page Special Issue are available for $20.00 each. Prepayment is required. Order reprints from the Ecological Society of America, Attention: Reprint Department, 1707 H Street, N.W., Suite 400, Washington, DC 20006. Costs for this Special Issue were defrayed by NSF grant DEB-0408432 to C. O. Webb and J. B. Losos. NCEAS funded a workshop on ‘‘Phylogenies and Community Ecology’’ in 2002, which a number of authors in this Special Issue attended.

S1 and Ackerly et al. attempt to detect which characters diverged earlier during the course of plant diversification. Knouft et al. use the new methods of GIS-based niche modeling to examine the evolution of multidimensional niches in a clade of lizards, by associating specimens’ collection locations with layers of environmental variation in space. Inferring evolutionary process from the pattern of character evolution we observe requires models of character change under known mechanisms. Unbounded continuous characters evolving under Brownian drift (slow relative to speciation rate) will tend to show conservatism (closely related species tend to be similar). Stabilizing selection will tend to increase that conservatism, while, conversely, a reduced number of potential character states, a long time between speciation events, and divergent selection will tend to increase convergence. Using various methods, the authors found generally more conservatism in ecological characters (e.g., Ackerly et al., Silvertown et al.) than convergence (e.g., Knouft et al.), with some studies finding no clear relationship of traits with relatedness (e.g., Agrawal and Fishbein). Overall, the results are consistent with the action of divergent selection in some systems, overlaid on a null expectation of some level of phylogenetic conservatism in all systems. The third major thread in these papers is closely linked to the second: ‘‘what is the phylogenetic relatedness of co-occurring taxa in communities, and what does this tell us about community assembly?’’ Authors used a number of methods to combine community lists with phylogenies to answer this question. Cavender-Bares et al. and Lovette and Hochachka correlated taxon co- occurrence rates (across many samples) with phylogenetic distance (e.g., with Mantel tests). Horner- Devine and Bohannan, Kembel and Hubbell, Weiblen et al., Silvertown et al., and Webb et al. tested the observed distribution of intra-sample, inter-taxon phylogenetic distances against null models of community assembly (so raising many of the perennial questions of community null models). Several authors pointed out that methods using inter-taxon phylogenetic distance rather than ancestral state reconstruction are less prone to bias introduced by the sampling of taxa that are very widely distributed on the tree of life. Several authors found that taxa in their communities were more closely related than expected, indicating a common role of habitat choice and evolutionarily conserved characters (e.g., Horner-Devine and Bohannan, Weiblen et al.). Cavender-Bares et al. and Kembel and Hubbell found cases where taxa were less closely related than expected. Taking the results of trait evolution and community phylogenetic structure together, the importance of community interactions does appear to be diminished, and a long-term regional view of taxa more justified. However, Lovette and Hochachka found both conservatism of habitat specialization in warblers at regional scales and evidence for competitive ‘‘repulsion’’ among close relatives at local sites. Because different combinations of trait evolution pattern and ecological interaction (competition vs. habitat choice) can give similar community phylogenetic structure, trait data, community data, and phylogenies are all needed for a full understanding of the evolution and assembly of communities. The discussion of the nature of communities and niche evolution is decades old; note that a similarly titled Special Feature appeared in this journal ten years ago. However, the vast number of species that have been sequenced, and for which phylogenies have been generated, means that ecologists can now often infer the phylogenetic relationships of their taxa from publications and databases without further systematics work. We hope that this Special Issue will inspire readers to take advantage of these opportunities, to phrase their questions in a more evolutionary way, and as Westoby anticipates, to participate in the new Natural History. —CAMPBELL O. WEBB Guest Editor Harvard University

—JONATHAN B. LOSOS Guest Editor Washington University

—ANURAG A. AGRAWAL Special Features Editor Cornell University

Key words: character evolution; community structure; competition; habitat ﬁltering; multidimensional niche; null models.

Ó 2006 by the Ecological Society of America Ecology, 87(7) Supplement, 2006, pp. S3–S13 Ó 2006 by the Ecological Society of America

EVOLUTIONARY DIVERSIFICATION AND THE ORIGIN OF THE DIVERSITY–ENVIRONMENT RELATIONSHIP

1 ROBERT E. RICKLEFS Department of Biology, University of Missouri–St. Louis, 8001 Natural Bridge Road, St. Louis, Missouri 63121-4499 USA

Abstract. Global patterns in species richness have resisted explanation since they first caught the attention of ecologists in the 1960s. The failure of ecology to fully integrate the diversity issue into its core of accepted wisdom derives from an inappropriate concept of community and the rejection of history and region as formative contexts for ecological systems. Traditionally, ecologists have held that the pervasive relationship between species richness and conditions of the physical environment reflects the influence of environment on the ability of populations to coexist locally. However, many ecologists now recognize that this relationship can also develop historically from the evolutionary diversification of lineages within and between ecological zones. To assess the relative roles of local ecological constraint vs. regional and historical unfolding of diversity–environment relationships, we must abandon localized concepts of the community and adopt historical (particularly phylogenetic) and geographic methods to evaluate the evolution of diversity within large regions and its influence on diversity at local scales. This integrated perspective opens new research directions for ecologists to explore the formation of species, adaptive diversification, and the adjustment of ecological distributions of species on regional scales. Key words: adaptation; community; diversity gradient; history; local determinism; phylogeny; saturation; speciation; species packing; species richness.

INTRODUCTION which the relative contributions of local and regional processes can be weighed, and sketch out some The number of species at all spatial scales varies implications of diversity patterns for our concepts of widely over the surface of the earth. Ecologists have ecological systems. sought explanations for patterns of diversity in the All biological systems have general properties, which varied expression of ecological processes under different are governed by the pervasive influences of thermody- local physical conditions of the environment. However, namics, evolutionary adaptation, and other ubiquitous patterns in diversity also can be explained plausibly as processes. They also have special properties, which the outcome of large-scale processes that control the reflect the unique history and present-day circumstances production and extinction of species within regions, of every species and location. These special properties which in turn influence the number of species in local form the foundation of systematics and biogeography. assemblages. These alternatives are not mutually ex- Ecologists traditionally have been concerned with clusive, and ecologists are beginning to join features of general properties of systems arising from universal both into a unified theory for the origin and main- processes with deterministic outcomes. Ideas about the tenance of patterns of diversity. The task is more distributions of organisms, population regulation, pop- difficult than it would seem, because it requires the ulation interactions, community succession, and ecosys- integration of dissimilar properties of biological systems: tem energetics have stemmed from such thinking deterministic properties that are molded by local (Kingsland 1985, McIntosh 1985, Brown et al. 2004). ecological processes, and the outcomes of evolutionary Global patterns of species richness occupy an uncertain and biogeographic processes that depend on unique position between the special and the general. Early features of the history and physiography of regions treatments regarded such patterns as the special out- (Ricklefs 2004, 2005a). Because local determinism has come of history and, therefore, outside the realm of been so prominent in ecological thinking, I first consider ecology (Wallace 1878, Matthew 1915, Willis 1922, how local determinism has achieved its current status, Fischer 1960). Tropical diversity was thought to reflect then briefly review some of the conflicting data that the greater age, area, and climatic stability of equatorial, weaken this central ecological paradigm, suggest ways in compared to temperate and boreal, environments, which allowed ample time and opportunity for the evolution of diverse forms of life (Dobzhansky 1950, Pianka 1966). Manuscript received 17 January 2005; revised 22 August 2005; accepted 7 September 2005. Corresponding Editor (ad THE RISE OF LOCAL DETERMINISM hoc): C. O. Webb. For reprints of this Special Issue, see footnote 1, p. S1. With the development of community ecology as a ma- 1 E-mail: [email protected] ture discipline in the 1960s, ecologists began to regard S3 S4 ROBERT E. RICKLEFS Ecology Special Issue diversity as a general feature of biological systems the flexible filling of niche space was too complicated to regulated locally by processes with deterministic out- be handled by theory. Eventually, local community comes (MacArthur 1965, 1972). Moreover, because local ecology was insulated from such external drivers as population interactions achieve equilibrium within tens colonization and regional species production. Many of generations, they were thought to be fast enough to ecologists accepted local saturation, and they reconciled override more ponderous regional and evolutionary differences between local and regional diversity by processes (Ricklefs 1989). These considerations led to a differential turnover of species between habitats (beta theoretical construct wherein population interactions diversity; Cody 1975). Thus, while recognizing the limited membership in a community to species that are influence of large-scale processes on regional diversity, ecologically compatible (MacArthur 1968, May 1975, most ecologists still regarded patterns of local diversity Case 1990, Morton et al. 1996). Accordingly, differences as the consequence of ecological sorting of species in the number of species between communities reflected available within a region (MacArthur 1965, Diamond the different outcomes of species interactions under 1975, Zobel 1997, Weiher and Keddy 1999). particular environmental conditions. This implied that diversity would be correlated with variation in the NEW CONCEPTS OF ECOLOGICAL COMMUNITIES physical environment, which has been borne out, to Recent excitement over Hubbell’s (2001) neutral greater or lesser degree, by empirical studies (e.g., theory of communities suggests that many ecologists Mittelbach et al. 2001, Hawkins et al. 2003b). were willing, after struggling for decades without Ironically, the rise of local determinism in community resolving the diversity problem, to entertain theories ecology occurred almost simultaneously with two other that discount ecological interactions and niche special- developments that contradicted its basic tenets. The first ization completely. Hubbell’s theory is historical and was the acceptance by most ecologists of an ‘‘open’’ geographical (geohistorical). Diversity depends solely on community structure (Gleason 1926), which is to say the area of suitable habitat within a region (that is, that communities lack boundaries and that locally proportional to the number of individuals in the coexisting species have more or less independent metacommunity) and the rate of species production, distributions over spatial and ecological gradients within assuming that systems have had sufficient time to attain regions (Whittaker 1967). The second was the coloniza- equilibrium. Species extinction is purely stochastic, tion–extinction steady state in MacArthur and Wilson’s depending only on the size of a population. The theory equilibrium theory of island biogeography (MacArthur includes ecology only in the sense that the total number and Wilson 1967), in which local (island) diversity is of individuals within the regional metacommunity is responsive to an external driver (colonization). fixed. Thus, all individuals compete on a homogeneous Referring to the number of species on islands of ecological landscape, but on equal footing irrespective of different size close to the source of colonization, their identity. As species richness within the metacom- MacArthur and Wilson (1963) introduced the idea of munity increases, average population size decreases, as saturation. In their meaning of the word, saturation was does the time to extinction. This balances species not a local property, but reflected the influence of the production to arrive at the equilibrium diversity. size of an area on sampling properties and rates of Although a purely neutral theory does not withstand extinction of populations. Abbott and Grant (1976) also scrutiny on a number of counts (Chave 2004), partic- used this concept of saturation to represent diversity ularly because neutral drift is too slow to account for the within a particular continental source area for colonists, development of ecological patterns (Ricklefs 2006a), a against which the diversity of islands could be measured. purely local, ecological theory of diversity also cannot These empirical concepts were transferred to local account for certain empirical patterns. These include communities through theoretical analyses of limiting correlations between local and regional diversity (Cor- similarity among species (e.g., MacArthur and Levins nell and Lawton 1992, Srivastava 1999), which contra- 1967), which led to the idea of species packing and its dict the idea of local saturation of species richness, and corollary that diversity (species coexistence) was con- incomplete convergence in diversity between areas of strained by the capacity of the local environment to similar environment in different regions with indepen- support interacting species. During the following de- dently evolved biotas (e.g., Latham and Ricklefs 1993a, cade, ecologists conducted comparative studies of com- Qian and Ricklefs 2000). munities largely in the context of species packing and the A geohistorical, evolutionary alternative to both local partitioning of niche space, presuming that environment determinism and neutral theory is the generation of constrained diversity (e.g., Pianka 1973, Cody 1974). gradients of species richness through diversification In fact, community theory sets no upper ‘‘saturated’’ within ancestral ecological zones of origin combined limit to diversity in a particular environment. Referring with occasional adaptive shifts associated with invasion to a community of competing species, MacArthur (1970) of new ecological zones (Farrell et al. 1992, Latham and emphasized that ‘‘... in a constant environment there is Ricklefs 1993b, Wiens and Donoghue 2004). The idea almost no limit to the number of species which can presumes that species are best adapted to the conditions improve the fit and hence be packed in ... .’’ However, in the ecological zone of origin of their lineage; July 2006 THE DIVERSITY–ENVIRONMENTAL RELATIONSHIP S5 transitions to other ecological zones require evolutionary change. This evolutionary model establishes gradients of diversity, producing greater species richness in environments that are older, more widespread, or less stressful. The idea originated with biogeographers (e.g., Darlington 1957, Axelrod 1966), but was slow to be adopted by ecologists. Terborgh (1973) was the first to articulate a comprehensive theory of community diversity that explicitly included historical and geographic influences on local species richness. Applied, for example, to the species richness of forest trees, the latitudinal gradient could be explained by the origination and diversification of most flowering plant lineages in the extensive tropics of the early Tertiary (Burnham and Johnson 2004, Davis et al. 2005), followed by diversification of some lineages across adaptive barriers into high-latitude frost zones (Latham and Ricklefs 1993b). FIG. 1. Evolutionary diversification of a clade within its This process is shown diagrammatically in Fig. 1. ecological zone of origin, with occasional adaptive shifts (stars) to different ecological zones. The adaptive shifts might be A second evolutionary alternative to local determi- difficult and occur infrequently, potentially establishing a nation is that rates of evolutionary proliferation of gradient in contemporary diversity that favors the ecological species (speciation minus extinction) are higher in zone of origin. Modified from Ricklefs (2005a); see also Wiens regions or ecological zones of high diversity (Farrell and Donoghue (2004). and Mitter 1993, Jablonski 1993, Cardillo 1999). This mechanism could be called a general process if certain the same predictions, which cannot then be used to ecological conditions, such as temperature, promoted or distinguish between them. For example, if species retarded proliferation (Rohde 1992, Allen et al. 2002), or richness were determined locally by ecological con- a special process if proliferation depended on unique straints on species packing, the phylogenetic history of physiography or geographic configurations of regions or the species in regions having different diversity might if extinction depended on unique history. For example, also be consistent with a diversification constraint. Qian and Ricklefs (2000) speculated that the high A simple classification of the mechanisms that diversity of plants in temperate eastern Asia could be influence diversity is presented in Table 1. These are related, in part, to the complex physiography of the divided into local and regional/historical processes, and region, which is both mountainous and features land the latter are further subdivided into the influences of masses (China mainland, Korean peninsula, and Japan) the ecological zone of origin, of environment on the rate that have alternately been connected and separated as of diversification, and of diversity itself in promoting or sea levels have risen and fallen during the Tertiary. retarding further diversification. Molecular phylogenetic analysis of plant clades distributed in eastern Asia and North America supports both Community saturation an older age (Asian taxa paraphyletic to North Taken to its extreme, local determinism predicts that American taxa) and more rapid diversification in eastern communities are saturated with species and that species Asia as underlying causes of diversity differences (Xiang richness is directly related to factors in the physical et al. 2004). Extinction also can play a role (Vermeij environment that determine the number of species that 1987, Jablonski 1991). For example, Latham and can coexist locally (Table 1: Panel A). Community Ricklefs (1993a) and Svenning (2003) concluded from saturation implies an upper limit to the number of comparisons of fossil and modern taxa that the species in a local assemblage, regardless of the diversity impoverished tree flora of Europe resulted from differ- within the surrounding region (Cody 1966). In principle, ential extinction of species within predominantly trop- this hypothesis can be confirmed in comparisons of ical and subtropical groups caused by cooling climates species assemblages in the same habitats between areas and glaciation during the late Tertiary. having different regional diversity, i.e., the principle of community convergence (e.g., Orians and Paine 1983, THE INFLUENCE OF LOCAL AND REGIONAL PROCESSES Schluter and Ricklefs 1993), particularly when local If one accepts the premise that local diversity diversity levels off with increasing regional diversity. represents a balance between the constraining influence Terborgh and Faaborg (1980) were the first to apply a of local population interactions and the augmenting saturation test explicitly, with a positive result. Most influence of regional species production, then one can subsequent studies have failed to find evidence for a hope to estimate only the relative balance of these hard upper limit to species number, but Srivastava factors in determining patterns of species richness. (1999) called attention to the shortcomings of such Moreover, general and special processes often make analyses, including inconsistent definitions of local and S6 ROBERT E. RICKLEFS Ecology Special Issue

TABLE 1. A classiﬁcation of inﬂuences on large-scale patterns in local diversity.

Mechanism Comments A) Local determinism Explanations based on local factors require explicit models of how the physical environment influences coexistence. a) Limiting similarity and saturation This extreme form of local determinism predicts community convergence, which is rejected in many comparative studies. b) Diversity increases resistance Ecological compression and release demonstrate the influence of population to invasion interactions on local community membership. B) Regional/historical processes These models predict diversity within larger regions, but also provide external drivers for local species richness. a) Ecological zone of origin Phylogenetic analysis permits the reconstruction of ancestral ecological positions within the environmental landscape. i) Age and area Phylogenetic analysis provides an estimate of relative age; area effects are apparent in contemporary biotas, but the relative roles of species production and extinction should be distinguished. ii) Adaptive diversification Zones of origin can be identified by phylogenetic analysis, and these should support the highest species richness regardless of the depth of the clade stem. b) Net rate of diversification Differences in rates of diversification can be seen in lineage-through-time plots and inferred, to some degree, from genetic distances between sister taxa. i) Physiography and history Assuming allopatric speciation, regional analyses of geographic heterogeneity at promote speciation appropriate scales would be informative, as would studies of incipient species formation (e.g., genetic differentiation of populations) ii) Climate change and catastrophes This can be judged primarily through analysis of fossil material and the geological cause extinction record, hence applications are limited to groups with good fossil records. iii) Diversity promotes or retards Diversity might be self-accelerating or self-limiting. The pattern is accessible diversification through analysis of lineages through time and analysis of biological and physical aspects of niche structure in contemporary biotas.

regional scales and lack of independence between determination commonly fall in the range of 60–70%,or regions. Ricklefs (2000) emphasized the balance between more (Hawkins et al. 2003a). However, such correlations local and regional factors, pointing out that increased might also be predicted by evolutionary theories of ‘‘regional’’ diversity among West Indian island avifau- species richness patterns, where diversity reflects either nas was accommodated equally by increases in local environmental history combined with evolutionary species richness and turnover of species between inertia, or the influence of environment on net species habitats. Consistent with this ecological flexibility, proliferation. introduced species apparently have not caused extinctions on islands through competitive exclusion (Sax et Unique history and geography al. 2002; but see Gurevitch and Padilla (2004)), and Local constraints predict convergence of local com- establishment of new colonists in the avifauna of the munity properties in similar environments regardless of Lesser Antilles appears to be independent of local the evolutionary and geographic history of a region, diversity (Table 1, [Panel B, row b(iii)]; Ricklefs and within which species richness might vary widely. Bermingham 2001). Over longer periods observable in Conversely, differences in species richness in the same the marine fossil record of the Paleozoic Era, for habitat between regions suggest that special factors example, diversity does appear to have constrained influence local assemblages. Such differences have been diversification (e.g., Foote 2000). noted frequently in community comparisons. Among the most conspicuous of these diversity anomalies in Local constraint plants, for example, are the difference in species richness Local constraint predicts that local species richness between mangrove communities in the Indo-West should be strongly correlated with environmental con- Pacific and the Atlantic/Caribbean regions (Duke ditions, irrespective of the mechanisms regulating 1992, Ricklefs and Latham 1993), the depauperate tree coexistence. Under local determinism, diversity patterns flora of Europe compared to eastern Asia (Latham and must ultimately be related to physical aspects of the Ricklefs 1993a), and the greater diversity within disjunct environment (e.g., Ricklefs 1977, Currie et al. 2004). genera of angiosperms in eastern Asia compared to Many studies have demonstrated strong correlations eastern North America (Qian and Ricklefs 1999). Such between local species richness, using various definitions diversity anomalies have been explained by differences of the scale of ‘‘local’’ (Rahbek and Graves 2001, Willis between regions in (a) the frequency of adaptive shifts of and Whittaker 2002), and climate (Currie 1991, O’Brien new lineages to the stressful mangrove environment, (b) 1998, Hawkins et al. 2003b) or other variables, such as extinction in the case of the European tree flora, and (c) habitat structure (Cody 1974) and productivity (Rosen- a combination of species production, species invasion zweig 1995, Mittelbach et al. 2001). Coefficients of from the tropics, and late-Tertiary extinction in the case July 2006 THE DIVERSITY–ENVIRONMENTAL RELATIONSHIP S7 of the disjunct temperate floras of Asia and North ation rate and the ages of lineages influence diversity America. Although special explanations for diversity within regions (e.g., Xiang et al. 2004). anomalies make good sense, they are suggested by the data themselves rather than emerging from the use of Ecological zone of origin data to test contrasting predictions. Special explanations Every lineage of organism has an ecological zone of are difficult to place in an experimental or hypothesis- origin (Table 1 [Panel B, row a]), which, under some testing framework (Francis and Currie 1998), and the circumstances, can be identified by tracing character anomalies themselves might reflect unmeasured differ- (i.e., ecological zone) evolution on a phylogenetic tree ences in local environments between regions (Pianka (e.g., Schluter et al. 1997). This is similar to inferring the 1975, Morton 1993, Ricklefs et al. 2004). geographic area of origin, which often can be ambiguous Can diversity anomalies be used other than to provide (Ronquist 1997, Brown and Lomolino 1998:Chapter 12, anecdotal support for the idea of special influences on Sanmartin et al. 2001). When an entire clade is restricted diversity? Some special geographic circumstances are to a particular ecological zone, one can infer that it repeated themes in many regions of the world. Among originated within that zone, even though this parsimo- terrestrial environments, for example, mountainous nious conclusion might be incorrect (Davis et al. 2005). areas coincide with diversity hotspots (e.g., Barthlott et When a clade is distributed over several ecological al. 1996, Orme et al. 2005), providing an opportunity to zones, identifying the origin in the absence of fossil test the consistency of some special relationships. Even evidence depends on the paraphyletic distribution of where these relationships are unique, geographic/histor- zones over the clade. ical models suggest mechanisms, such as enhancement of allopatric speciation or restriction of evolutionary Diversification across adaptive barriers diversification, which can be assessed in multiple, Some of the strongest diversity gradients are clearly independently evolving lineages. Thus, although empiri- associated with particular environmental stressors (Ta- cal observations might not provide statistical support ble 1 [Panel B, row a(ii)]), including high salt and for a particular historical scenario, one might obtain substrate anoxia in the case of mangrove vegetation and statistically valid appraisals of mechanisms postulated to freezing in the case of temperate vegetation. The roughly be at work within a unique geohistorical framework. 20 present-day lineages of mangrove trees and shrubs were independently derived from terrestrial lineages of Speciation rate plants over a period of more than 60 million years The role of speciation rate in creating patterns of (Ricklefs and Latham 1993, Ellison et al. 1999). This diversity (Table 1, [Panel B, row b]) has received suggests that the evolutionary transition from terrestrial considerable attention, particularly with respect to to mangrove environments is difficult and that this identifying the influence of ‘‘key innovations’’ on adaptive barrier is crossed infrequently. The same is diversification (Slowinski and Guyer 1993, Heard and likely true of evolutionary transitions from mesic Hauser 1995, Bennett and Owens 2002). Among the tropical to temperate habitats (Terborgh 1973). Phylo- more successful of these tests have been the association genetic analysis reveals that the lineages of trees in north of rapid diversification in insects with the switch to temperate latitudes are mostly nested within clades herbivorous diets and the association of rapid diversi- having deep tropical origins (Ricklefs 2005a). Indeed, fication in plants with the evolution of certain defenses more than half the families of flowering plants are against herbivory (Farrell and Mitter 1994). The restricted to tropical latitudes and evidently have not physiography of particular regions, such as eastern been able to cross the adaptive barrier into temperate Asia, might promote species production. General latitudes (Ricklefs and Renner 1994). During most of characteristics of the environment, such as the benign the early evolution and diversification of the flowering nature of tropical climates, could also influence speci- plants, the planet’s environments were predominately ation rate (Dobzhansky 1950, Schemske 2002). Several tropical (Behrensmeyer et al. 1992, Graham 1999). authors have suggested that thermal energy can accel- Models of the establishment of regional patterns of erate speciation (e.g., Rohde 1992, Allen et al. 2002). species richness, based on origins vs. rates of prolifer- Attempts to test hypotheses on the rate of speciation ation, can be distinguished by phylogenetic analysis have focused on comparisons of sister (same-age) clades (compare Figs. 1 and 2). Whether either of these in tropical and temperate regions (Farrell and Mitter evolutionary models were to shape gradients in local 1993, Cardillo 1999, Cardillo et al. 2005, Ricklefs 2005b, diversity, in contrast to merely reflecting the gradient in 2006b), but these have been relatively inconclusive numbers of contemporary coexisting species permitted because suitable data are difficult to assemble. Such by ecological interactions, is more difficult to resolve. comparisons depend on well-supported phylogenies. That is, all species have an evolutionary history, Because these are rapidly increasing in number, testing including relationships to other lineages, and one can the effect of various environmental or regional factors reconstruct phylogenies for the members of a local on rates of species production will become more community regardless of the mechanisms that control commonplace and will show whether and how speci- local diversity. A role for adaptive barriers can be S8 ROBERT E. RICKLEFS Ecology Special Issue

FIG. 2. When species proliferate more rapidly in a novel environment than in the ecological zone of origin, diversity need not coincide with the ancestral environment. In this example, the zone of origin can be identified through phylogenetic analysis, because species within the ancestral zone are paraphyletic with respect to species in the zone of highest diversity. inferred from the partitioning of evolutionary lineages adaptation are slow compared to local ecological between ecological zones (Webb 2000, Webb et al. dynamics. However, when the interactions between 2002). If species can cross between these zones easily, species play out within regions rather than local ecological zone itself would be labile, varying at a communities, general processes can adjust ecological shallow depth within a phylogeny, as in the case of oak and geographic distributions of species so that all species across a moisture gradient in Florida (Cavender- populations come into demographic balance within the Bares et al. 2004) or evolutionary transitions to chapar- region (Ricklefs 2004). Ironically, this deterministic ral environments in western North America (Ackerly outcome is independent of special aspects of regions 2004). However, if these adaptive shifts were difficult, as and lineages, including the number of species that have in the case of entering the mangrove environment, been generated within a region. As more species are ecological zone would appear to be a conservative trait. added, ecological distributions are compressed, beta For example, the four genera and perhaps 17 species of diversity (species turnover with respect to ecology and mangrove Rhizophoraceae represent a single lineage distance) increases, and equilibrium is maintained that entered the mangrove environment early in the (Terborgh 1973). This concept requires that ecologists evolution of the family and diversified there without any consider populations as regional ecological entities made lineages crossing back to the terrestrial ecological zone integral by the movements of individuals (Lennon et al. (Schwarzbach and Ricklefs 2000). 1997, Ricklefs 2004, Case et al. 2005, Holt et al. 2005). Evolutionary and geographic history—special com- PROSPECTS ponents of ecological systems—can be revealed through Ecological regions of high diversity and ecological phylogenetic analysis, among other approaches, which regions of origin likely coincide, although rates of line- provides insight into the development of diversity age proliferation also appear to differ between regions patterns and unique aspects of biological communities and might produce a contradictory pattern (Fig. 2). In in different regions. Speciation and extinction also general, ecological and evolutionary models for the reflect the intimate connections between ecology, geog- origin and maintenance of diversity gradients cannot be raphy, and evolution. Although many areas of ecolog- distinguished by examining patterns of diversity. Be- ical inquiry are independent of evolution and history, cause of this, ecologists must expand their inquiries to interactions between species played out within a large include evolutionary hypotheses (Webb et al. 2002, regional context bring ecological and evolutionary Ackerly 2004), and they must develop mechanistic, processes onto a continuum of scale that intimately testable, ecological models that connect diversity to the links local ecology, history, and geography. Within this physical environment (Currie et al. 2004). regional context, ecologists can characterize species Special explanations for species richness require that distributions, including sampling on a variety of scales special processes have strong enough influence to leave a that comprise local individual movements, population distinctive imprint on ecological pattern. Evolution and interactions, habitat heterogeneity, and regional pro- July 2006 THE DIVERSITY–ENVIRONMENTAL RELATIONSHIP S9 cesses of population subdivision and species formation 2) The history of distribution of clades over environ- (Rahbek 1997, Rahbek and Graves 2001). Islands, mental gradients involves adaptive changes and may particularly archipelagoes, will continue to be important constrain the diversity of species. Accordingly, it is laboratories for studying mechanisms of species origi- important to examine patterns of species richness across nation (Grant 1998, Mayr and Diamond 2001) and the environmental gradients in a phylogenetic framework. response of ecological distributions to the pressure of The general context for this is presented in Figs. 1 and 2. regional diversity (Cox and Ricklefs 1977). Analyses of Ecologists should focus on strong barriers (e.g., frost the niche structure of assemblages, particularly by tolerance, salinity gradients) as well as less imposing defining the multivariate dimensionality of niche axes temperature and moisture gradients within regions from ecological, morphological, and life history data (Ackerly 2004, Cavender-Bares et al. 2004). Such studies (Ricklefs and Miles 1994), will assess the contribution of should seek factors that influence multiple lineages and niche space to diversification and the complementary that presumably impose the strongest constraints. These contribution of niche diversification to the evolutionary analyses of habitat shifts should be based on well- development of species assemblages. Most of these supported phylogenies, preferably in which branch techniques are familiar to ecologists; new directions for points can be dated by fossils or calibrations developed the future will arise from their application in a novel for other lineages (e.g., Kishino et al. 2001, Renner and framework. Meyer 2001) to relate adaptive shifts to climate or Within this framework, I can make several specific geographic change, and they must also use proper recommendations. Of course, this list reflects my own estimates of ancestral environments (Schluter et al. perspective and would be broadened considerably by 1997). Although this approach is primarily descriptive others. First, it is important to understand that the scale rather than hypothesis testing, it can provide support for of analysis must be appropriate to the scale of the the prediction from adaptive constraint that ancestral process in both time and space. Thus, if one accepts the lineages might change abruptly at a deep level across strong environmental gradients (Westoby 2006). premise that regional and historical processes can 3) Building upon the analysis of population distribu- influence the development of local assemblages of tions within regions and phylogenetic analysis across species, then analyses of patterns of local diversity must environmental gradients, it seems reasonable to examine include regional distribution and both environmental local assemblages and distributions of species across and evolutionary history. These might be accommo- particularly important gradients of diversity. Among dated, in part, in the following ways. these, one that has been important in development of 1) It is essential that ecologists abandon the idea of the ideas about species richness patterns is the difference in local community. Interactions occur between popula- tree species diversity between tropical and temperate tions over large regions, and we should therefore environments. The transitions between these ends of an characterize the distributions of species on the geo- environmental continuum for broad-leaved trees are graphic and ecological gradients over which they interact located in areas (eastern Mexico, southern China) for (Case et al. 2005). What ecologists have traditionally which few floristic data exist, but where we might expect regarded as a ‘‘community’’ comprises the populations the greatest information concerning diversity gradients. that co-occur together at a particular point in these Although altitudinal gradients do not have the same regional continua of conditions. Whittaker (1967) and seasonal dimension as latitudinal gradients, they are also others used this approach in gradient analysis of informative to the extent that species turn over in vegetation. Sampling is conducted in plots along parallel fashion. environmental gradients, and species populations can 4) Several studies have examined the depth of the be characterized by their mean position, dispersion, and clades that comprise a community as a way to assess the modal abundance along these gradients. When gradients relationship between the ages of lineages and their are made comparable between regions, differences in the diversity. Simply put, if the net rate of diversification overlap and partitioning of species along these gradients (speciation minus extinction) is homogeneous, older can be compared as an approach to understanding how lineages will leave more descendants. That is, the population responses accommodate variation in region logarithm of the number of species is a function of the diversity (e.g., Nekola and White 1999, Qian et al. 2005). net rate of diversification and time. Ricklefs and Schluter Although this will be straightforward in principle, it may (1993) showed that the clades that comprised avian be difficult in practice because ecologists typically assemblages in a tropical locality in Panama were consider too few large-scale gradients within regions to roughly twice as old as the clades that made up an separate hundreds or thousands of species. Ordination assemblage in Illinois, based on field data in Karr (Karr and canonical correspondence analysis typically explain 1971) and lineage ages from Sibley and Ahlquist (1990). a modest proportion of the variance in species distribu- The implication is that time is an important factor in tions, leaving the rest to biological complexities in the both regional and local diversity. Hawkins (2005) has environment and chance, both of which have tradition- provided a more comprehensive analysis that shows the ally been ignored by ecologists (cf. Condit et al. 2002). same pattern in the birds of Australia, and Ricklefs S10 ROBERT E. RICKLEFS Ecology Special Issue

(2005a) confirmed the conventional botanical wisdom 7) Finally, extinction can play an important role in the (Judd et al. 1994) that temperate trees belong to clades establishment of patterns of diversity. Where an that are nested within deeper clades of tropical lineages. adequate fossil record exists, one has a direct estimate This topology is illustrated in Fig. 1, where the species in of diversity, at some resolution, through time. The the more diverse ecological region belong to clades that extinction of many clades of animals at the Cretaceous– have diversified within that region for longer periods. Tertiary boundary, as well as the disappearance of many 5) A second facet of the historical dimension is the rate plants from Europe during the late-Tertiary period of of diversification of species. All other things being equal, climate cooling (Sauer 1988, Latham and Ricklefs diversity increases as a function of the rate of 1993b, Svenning 2003), provide instructive examples of diversification and time (Ricklefs 2006b). Thus, faster the importance of this factor. A large literature in diversification leads to more species over a given period. paleontology addresses rate of extinction and its Although few studies have made this comparison to relationship to diversification, environmental changes, date, and these have been inconclusive, the increasing catastrophic events, and changes in the configurations of availability of phylogenetic reconstructions will change landmasses and ocean basins (Jablonski 1989, 1991, this prospect rapidly. Appropriate comparisons include 1993, Vermeij 1991, Jackson et al. 1993). Unfortunately, sister clades that occur in different regions or environ- the fossil record is rarely resolved to the taxonomic level ments (Farrell and Mitter 1993, Cardillo 1999), which, of species and hardly exists for many groups. This by definition, are of equal age, or samples of clades from should not prevent those of us interested in living forms different regions or environments for which age and from examining what is known of the fossil history of a number of taxa are known (e.g., Cardillo et al. 2005, group. At some level of resolution, this will provide at Ricklefs 2005b). Another approach is the lineage- least an envelope around possible historical scenarios. through-time plot (Nee et al. 1992, Harvey et al. 1994), Where fossil data are not available, information can be whose slope can be used, with well-sampled phylogenetic extracted from models of lineage diversification (e.g., reconstructions of large clades, to estimate both Magalloń and Sanderson 2001, Ricklefs 2006a) and speciation and extinction rates (e.g., Ricklefs 2006a). extinction inferred, for example, from gaps in the Another index to the rate of species proliferation is the distribution of species through archipelagoes (Ricklefs genetic divergence between sister species, which in and Cox 1972, Ricklefs and Bermingham 1999). general decreases as the rate of speciation increases The better we develop the space and time contexts of and extinction decreases. In all aspects of analyses that ecological systems, the more we shall appreciate the address species properties, results depend on the way in variety of factors that have influenced their composition which species are defined, which should be as uniform as and diversity. We should view populations as evolved possible across comparisons. entities, with unique histories and adaptations, that are 6) Speciation as a process contributing to regional distributed geographically according to their tolerance diversity can be examined phylogeographically by search- of ecological conditions and interactions with other ing for incipient speciation among geographically sepa- populations, and, within the limitations of dispersal, rated populations. Incipient species might be recognized over regional landscapes. Inevitable consequences of by large genetic differences between populations (Avise these processes are the co-occurrence of a unique 2000), but, in any event, these can be compared easily assemblage of species at any particular point and geographic patterns in the number of species observed among regions in terms of the number and geographic over many such points over the surface of the earth. distribution of populations or subpopulations at a particular phylogenetic depth (e.g., Brumfield and Cap- ACKNOWLEDGMENTS parella 1996, Bates et al. 1999, Weir and Schluter 2004). I am grateful to Cam Webb for discussion, encouragement, This type of analysis could identify regions of rapid and helpful comments on the manuscript. The National population differentiation that might lead to high rates of Geographic Society, Smithsonian Institution, National Science species production. Diversity is built up locally only when Foundation, and the University of Missouri Board of Curators have supported research related to this paper. geographically isolated, evolutionarily independent populations achieve secondary sympatry. Thus, comparison LITERATURE CITED of sister taxa in allopatry and sympatry (assuming an Abbott, I., and P. R. Grant. 1976. Nonequilibrial bird faunas allopatric model of species formation), would permit an on islands. American Naturalist 110:507–528. analysis of the time required for the local accumulation of Ackerly, D. D. 2004. Adaptation, niche conservatism, and convergence: comparative studies of leaf evolution in the species and whether the ecological differentiation that California chaparral. American Naturalist 163:654–671. permits coexistence evolves in allopatry, or primarily by Allen, A. P., J. H. Brown, and J. F. Gillooly. 2002. 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Ecology, 87(7) Supplement, 2006, pp. S14–S28 Ó 2006 by the Ecological Society of America

SIMULTANEOUS EFFECTS OF PHYLOGENETIC NICHE CONSERVATISM AND COMPETITION ON AVIAN COMMUNITY STRUCTURE

1 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Laboratory of Ornithology, Cornell University, 159 Sapsucker Woods Road, Ithaca, New York 14850 USA

Abstract. We currently have only a partial understanding of how phylogenetic relationships relate to patterns of community structure, in part because, for most groups of organisms, we do not know the extent to which ecological similarity results from common ancestry. Associations between phylogenetic relatedness and local community structure are particularly interesting for groups in which many species that span a gradient of phylogenetic divergence occur in potential sympatry. We explored the relationship between evolutionary relatedness and current species co-occurrence among the North American wood-warblers (Aves: Parulidae), a group of songbirds known both for its species diversity and for exhibiting high levels of sympatry at breeding sites. Species co-occurrences were derived from North American Breeding Bird Survey transects comprising 160 000 census points distributed across North America. The nested point-within-transect structure of this survey provides an unusual opportunity to remove larger-scale geographical effects on local community composition and thereby consider patterns of co-occurrence only among regionally sympatric pairs of species. We indexed evolutionary relatedness among all pairs of taxa by genetic distances based on long mitochondrial DNA protein-coding sequences. Most regionally sympatric taxon pairs rarely co-occur at local sites, and the most closely related never exhibit high local co- occurrences, as predicted if past or present competitive effects are strongest for these recently separated lineages. Quantile regression shows that, for a subset of taxa, local co-occurrence does increase with time since common ancestry, and that this apparent relaxation of competitive exclusion is strongest for distantly related species that have differentiated in fundamental ecological and behavioral traits, such as terrestrial vs. arboreal foraging. Comparisons against a null model of species co-occurrence further demonstrate that these patterns occur against a background of phylogenetic niche conservatism: across all phylogenetic distances, sympatric species co-occurred at higher rates than expected by chance, a pattern that might stem from a tendency by these species to show conservatism in their selection of similar general habitat types. Considered in concert, these analyses suggest the simultaneous mediation of local community structure by the ecological similarity of closely related species and by trait divergence among a subset of more distant lineages. Key words: community structure; competition; conservation; niche phylogeny; Parulidae; phylogenetic; wood-warbler.

INTRODUCTION the extent to which this ecological stasis results from the The connection between phylogenetic similarity and inheritance of niche-related traits from a common community composition is an important but largely ancestor is termed ‘‘phylogenetic niche conservation’’ (e.g., Wiens and Graham 2005). Research on phyloge- unresolved issue in evolutionary ecology. Because closely netic niche conservatism has been recently invigorated by related species will usually share many ecological traits the growing availability of robust phylogenetic hypoth- via common ancestry, evolutionary relationships are eses based on data independent from ecological traits, likely to be associated with patterns of community but few generalizable trends are apparent across assembly (Darwin 1859, Lack 1971, Ricklefs and Schluter studies. The present literature suggests that phyloge- 1993), but these phylogenetic effects might inﬂuence netic niche conservatism is detectable in some but not species coexistence in several ways. The general observa- all communities of organisms. Furthermore, its magni- tion that closely related organisms tend to occupy similar tude differs depending on the ecological traits under habitats and often use similar environmental resources comparison and additional factors, such as the degree long predates the founding of ecology as a science, and of ecological interaction among related species (Losos 1996, McPeek and Miller 1996, Peterson et al. 1999, Manuscript received 28 January 2005; revised 6 September Prinzing et al. 2001, Webb et al. 2002, Ackerly 2003, 2005; accepted 7 September 2005; ﬁnal version received 5 Losos et al. 2003, Anderson et al. 2004, Cavender-Bares October 2005. Corresponding Editor (ad hoc): J. B. Losos. For reprints of this Special Issue, see footnote 1, p. S1. et al. 2004, 2006, Wiens and Graham 2005, Kembel and 1 E-mail: [email protected] Hubbel 2006, Knouft et al. 2006, Weiblen et al. 2006). S14 July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S15

Competition among related species is likely one of the different years (Holmes et al. 1996, Cilimburg et al. principal forces causing the erosion of phylogenetic 2002), wood-warblers redistribute themselves across niche conservation: for species that interact ecologically, local habitats annually. This combination of large the initial ecological similarity that results from common population sizes, repeated historical displacement of ancestry will likely increase competition, potentially geographic ranges, and high individual dispersal means leading over time either to competitive exclusion or that most wood-warbler individuals breed at locations ecological differentiation (Webb et al. 2002). This where other wood-warbler species are present. process is most evident in adaptive radiations, in which As documented by Robert MacArthur in his classic groups of sympatric species have diversified extensively study of five sympatric ‘‘spruce woods’ warblers’’ with concomitant differentiation in traits related to their (MacArthur 1958), at the local scale co-occurring use of ecological resources (Losos and Miles 2002). In songbird species show differentiation in their spatial such situations, divergent selection that results from foraging niches and other traits (e.g., Holmes et al. 1979, competition could overwhelm any underlying retention Morse 1989, Suhonen et al. 1994). In a MacArthurian of niche conservatism, as found by Losos et al. (2003) framework, behavioral and ecological differentiation for a highly diverse community of Cuban Anolis lizards, reduce interspecific competition and permit related within which phylogenetic relationships explained only a species to co-occur. However, whether local niche small amount of the among-species ecological variation. differentiation explains broad spatial patterns of species This is perhaps an extreme example of a general trend: co-occurrence is controversial because the species most groups of closely related and ecologically interact- composition of wood-warbler communities varies across ing species will share some traits by common ancestry sites and years (Wiens 1989). and differ in other traits, owing to divergent selection A principal reason why the effect of evolutionary and other processes of evolutionary differentiation. relatedness on community structure remains largely An interesting intermediate situation involves groups unexplored is that some fundamental tests of the in which the species composition of local communities relationship require information from different spatial varies substantially in time and space, as these taxa are scales (Anderson et al. 2004, Cavender-Bares et al. individually involved in many potential interactions. In 2006): the competitive ecological interactions that can the absence of more stable associations between drive local patterns of community structure occur at a particular species that result in tight coevolutionary fine local scale best studied at points or in small study responses, species in highly variable communities might plots, yet these interactions must simultaneously be be more likely to assort themselves along relatively few integrated across the much broader geographic distri- axes of dissimilarity and therefore retain a larger butions of potentially co-occurring taxa. Most previous signature of phylogenetic niche conservation. In addi- studies of phylogenetic patterns in community structure tion, groups of organisms with high diversity, geo- have concentrated on interactions at one of these scales graphic distributions that have been labile through (e.g., Webb 2000, Silvertown et al. 2001, Losos et al. evolutionary time, and high individual dispersal are 2003, Gillespie 2004, Kembel and Hubbell 2006); particularly appropriate models for exploring how however, unique avian censuses organized by the North phylogenetic relationships relate to the balance between American Breeding Bird Survey (BBS) program (Sauer niche conservation and ecological divergence, because et al. 2003) allow us to synthesize complementary these attributes will diminish the degree of biogeo- information at both levels. Distinguishing between these graphic determinism in community composition by alternatives requires separating the biogeographic or bringing together many pairs of taxa that would other historical factors that constrain the pool of otherwise be allopatric. The diverse North American candidate species from the ecological factors that cause Parulidae fit these criteria, as nearly all wood-warbler competitive exclusion or facilitate co-occurrence species have persisted as independent lineages for at least (Ricklefs 1987, 2006). Underlying historical factors several million years (see Plate 1; Lovette and Berming- unrelated to direct competition include the fact that ham 1999, 2002). During their tenures in North lineages that have speciated in allopatry might remain America, the continental landscape has been trans- separated by the same physiographic features that formed many times by glacial and climate cycles and caused their initial isolation and hence never come into consequent broad-scale shifts in habitat distributions ecological contact. Even pairs of species that do occur in (Graham 1999, Klicka and Zink 1999, Johnson and sympatry will usually have geographic distributions that Cicero 2004, Weir and Schluter 2004, Lovette 2005). do not perfectly overlap. Current ecological interactions Nearly all of the approximately 6.0 3 108 individual are thus limited to a part of each species’ overall range. North American wood-warblers (Rich et al. 2004) also The fact that each species has a unique geographic migrate annually, and many studies of marked individ- distribution that varies in overlap with those of other uals (summarized in Poole and Gill [2004]) have shown species is a general challenge in comparing ecological co- that natal philopatry is low, at least at the fine local scale occurrences across many different pairs of species, as an of intensively monitored study plots. Resulting from this absence of species’ overlap at a given point could result natal dispersal and movements of adults among sites in from being outside one species’ geographic range, or S16 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Ecology Special Issue

PLATE 1. The Prairie Warbler (Dendroica discolor) is one of 42 common species of North American wood-warblers that frequently breed in sympatry with closely related species. Photo credit: Marie P. Read. more interestingly from ecological processes, such as competition among closely related species limits their differential habitat selection or competitive exclusion local ecological co-occurrence. In further analyses, we (Stone et al. 1996, Ackerly et al. 2006, Silvertown et al. explore whether these patterns are influenced by a 2006). The spatial structure of the BBS (i.e., many fundamental attribute of warbler behavior that leads to census points replicated along short transects) is strong spatial niche segregation, the species-specific particularly useful for our purposes because it allows tendency to forage terrestrially or arboreally. We also us to remove the confounding effects of these larger compare patterns of co-occurrence at the local spatial scale differences in total geographic range from our scale defined by census points, with the regional-scale measures of local co-occurrence. patterns defined by entire census transects. Unlike most Here, we use BBS census information in combination previous studies of phylogenetic effects on community with DNA-based genetic distances to assess whether structure, the structure of our data sets requires that we phylogenetic relatedness among regionally sympatric wood-warblers is positively or negatively correlated with compare matrices of genetic and ecological distances; co-occurrence at the same points in space, the birds’ our comparisons therefore do not require ancestral state local breeding sites. A positive relationship (i.e., related reconstructions and thereby minimize the importance of species co-occurring at higher than expected rates) the underlying assumption that we have identified the would suggest that local community composition is correct phylogenetic tree topology. Instead, we use a mediated in part by phylogenetic niche conservatism of null-model approach to identify and explore associa- habitat or habitat-linked aspects of niches, whereas a tions between phylogenetic relatedness and overlap in negative relationship would suggest that past or present species’ use of local breeding sites. July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S17

FIG. 1. Locations of Breeding Bird Survey (BBS) transects. Colors indicate the total number of wood-warbler species on each transect. Inset histograms depict the distributions of species richness on the .4000 transects and at the .162 000 census points within transects.

METHODS least partially overlapping territories. To derive a Breeding bird survey co-occurrences measure of local sympatry, we considered only BBS transects on which both members of each pair of species Breeding Bird Survey (BBS) censuses are run during were detected, as this physical proximity indicates that the breeding season by experienced volunteers who both species are members of the pool of taxa from which conduct standardized 3-minute counts of all birds heard we drew the local community at each census point. For or seen at 50 equally spaced locations along a 39.4-km each point observation of species A, we determined the (25-mile) transect (Sauer et al. 2003). Most transects are probability that wood-warbler species B was detected at recensused annually, and data spanning 1997–2003 are that same census point, generating an index of dissim- available for the species detected at each census point, as ilarity in co-occurrence that spans 0 (complete co- well as the summed observations for each transect. The occurrence) to 1 (no co-occurrence). To account for resulting 162 800 census points (Fig. 1) provide a robust differences in prevalence among species, the matrix of representation of all but the most geographically co-occurrences also included the reciprocal calculation restricted or peripheral North American wood-warblers. for each species pair (i.e., probability of detecting species Here we define the wood-warblers detected at a single A at points where species B was found). These values census point as co-occurring in local sympatry, as these provide a continent-wide assessment of species co- individuals are certainly within auditory detection occurrences measured only at the fine spatial scale most distance of one another and are likely to occupy at relevant to current ecological interactions among S18 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Ecology Special Issue species. Scaling these values such that higher numbers assume that among-year detection probability contains indicate lower co-occurrence facilitates comparisons information about within-year detection probability, the with the corresponding pairwise genetic distances, in latter being the detection probability of interest. which higher values indicate more ancient common Values of the detection index spanned 0.43–0.62 for ancestry. different species, with a median of 0.52 and a mean value We have contrasted these patterns of local co- of 0.53. Thus, values of the index were roughly occurrence with patterns of regional co-occurrence, in symmetrically distributed, and most species detection order to determine whether patterns found in local co- rates were at intermediate values: the interquartile range occurrence are the result of phenomena that were not spanned 0.50–0.56. While our index of detection happening at larger scales. Regional co-occurrence was probability is biased high relative to true detection defined as existing when both members of a species pair probability, due to the use of data for which each species were reported at least once on an individual 50-point had a minimum probability of 0.4 of being detected at transect in a given year, regardless of whether the two each individual site, this bias should be roughly equal species were reported at the same point. Taken across all for all species. BBS transects, regional co-occurrence was given as a value ranging from 0 (species B is always reported on a Spatial foraging niche characters transect that also reported species A) to 1 (species B is Co-occurring songbirds usually vary in traits that never reported on a transect that has species A). include foraging height, foraging substrate, and nest We base both local and regional co-occurrence indices location (e.g., MacArthur 1958, Lack 1971, Robinson on reported detections of warbler species, and each and Holmes 1982, Morse 1989, Richman and Price detection is a combined result of the presence of a bird 1992, Martin 1998), suggesting that within-site niche species at a site and the human detection of the individual partitioning is pervasive in these communities. There is bird(s). Because the detection data result from both a also strong evidence that some closely related wood- biological process and a methodological filter (detection warblers compete antagonistically in sympatry (Martin by observers), it is important to know whether the and Martin 2001). MacArthur’s 1958 paper spawned a patterns found reflect the biological process and not a large number of subsequent studies of wood-warbler methodological artifact. To our knowledge, there has niche segregation in traits such as foraging behavior, been no comprehensive attempt to estimate the detection habitat strata, nest locations, and prey types, but probabilities of all of the warbler species examined in this methodological differences among studies in different study. While we cannot calculate these detection local communities make it difficult to integrate these probabilities in a fully rigorous manner (e.g., MacKenzie measures into a common index of niche differentiation. et al. 2002) with the data at hand, we can calculate indices Instead, we coded a fundamental and unambiguously of detection probability that allow us to explore some categorizing attribute of the foraging niche of wood- potential biases. For each species of wood-warbler, we warblers, designating each species as either terrestrial randomly selected a single point on each BBS transect (mean foraging height ,1 m; 10 species) or arboreal that had reported the species on at least two points in at (mean height .1 m; 33 species) based on quantitative least one year; the actual median number of points at foraging studies conducted during the breeding season which a species was reported ranging from 2 (for one (Poole and Gill 2004). Each pair of taxa could therefore species only) to 11 across all species. The transect had to comprise species that forage primarily in similar be censused for at least five years, and transects censused (terrestrial–terrestrial and arboreal–arboreal pairs) or for longer had one or two years of data randomly separated (terrestrial–arboreal) strata. This categoriza- discarded to leave exactly five years of available data. tion allowed us to determine whether species pairs that Using a standardized number of years’ data avoided are predicted to interact more intensively (because they inducing variation in the detection index due to differ- occur in the same stratum; MacArthur 1958, Morse ences among species in the number of years that transects 1989:293, Wiens 1989) show a different association were censused. The randomly selected census point between phylogenetic relationship and co-occurrence within the transect had to have reported the target than do species pairs with a higher degree of spatial species in at least two separate years. Our rationale is that separation. we are purposefully selecting transects in regions in which a given species was likely to present, as well as Taxon sampling specific census points at which habitat was suitable and We included 43 Parulidae species that breed within the the species potentially present every year. The detection BBS region in the continental United States and Canada, index for each species was the proportion of point-years, excluding only six rare or geographically peripheral summed across all selected points, on which that species species that were recorded on few (10) BBS transects; was detected. The values of the detection indices used all included species were recorded on 41 transects. here are the median values from 1000 iterations of the Excluded species were Colima Warbler (Vermivora process of randomly selecting a single census point per crissalis), Tropical Parula (Parula pitiayumi), Golden- transect. In calculating these indices of detection, we cheeked Warbler (Dendroica chrysoparia), Kirtland’s July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S19

Warbler (D. kirtlandii), Red-faced Warbler (Cardellina alternative distance metrics were highly congruent (see rubrifrons), and Painted Redstart (Myioborus pictus). We Results and Discussion), the figures printed here illustrate excluded two additional species, the Yellow-breasted only the ML distance-based comparisons; the Appendix Chat (Icteria virens) and the Olive Warbler (Peucedra- depicts the alternative figures based on the ultrametric mus taeniatus), because, although these morphologically distance matrix. aberrant lineages have traditionally been placed in the We reconstructed phylogenetic relationships among Parulidae, more recent phylogenetic analyses (Sibley and the 43 species using the same sequences employed for the Ahlquist 1990, Klicka et al. 2000, Lovette and Berming- genetic distance calculations. We generated these phy- ham 2002) have shown unambiguously that they fall logenetic analyses via the Bayesian MCMC approach outside the Parulidae and that they are more closely implemented in MrBayes 3.0b4 (Huelsenbeck and related to species in other families. Ronquist 2001) under the GTR-gþI model of sequence evolution. The search was run for 5 3 106 generations Genetic distances and sampled every 2500 generations; the initial 1000 As indices of phylogenetic similarity, we generated samples were discarded as burn-in. An important two matrices of genetic distances among the 43 wood- assumption in our use of mtDNA-based distances as a warbler species based on the complete DNA sequences measure of species relationships is that the mtDNA gene of the mitochondrially encoded cytochrome oxidase I, tree reflects the overall organismal ‘‘species tree’’ for cytochrome oxidase II, NADH dehydrogenase II, these taxa. This assumption would be violated if past ATPase 6, and ATPase 8 genes (4116 nucleotides/taxon; hybridization resulted in interspecific mitochondrial GenBank accession numbers AY650182–AY650224). transfer (introgression), as has been documented in a Noncoding spacer regions and tRNA sequences situated few avian groups in which closely allied species breed in between these coding genes were sequenced, but they sympatry (e.g., Gala´pagos finches; Sato et al. 1999), or were excluded from distance calculations owing to their between species with active hybrid zones. In the paruline different underlying pattern of molecular evolution. warblers several lines of evidence—including long, These sequences are substantially longer than the species-specific mtDNA lineages (Lovette and Berming- mitochondrial alignments typically generated for spe- ham 1999, 2002) and the very high congruence between cies-level avian phylogenetics; by basing our distance the mtDNA gene tree and independent trees from DNA metric on this robust mtDNA data set, we minimize the sequences from six unlinked nuclear loci (I. J. Lovette, stochastic error in our estimates of pairwise distances. unpublished data)—suggest that the mitochondrial tree is Laboratory methods used to generate these sequences not highly biased by past introgression. The relative have been described elsewhere (e.g., Lovette 2004). constancy of mitochondrial substitution rates is also We calculated the first matrix of pairwise distances apparent in this phylogram, in which there are no using the maximum likelihood (ML) method imple- conspicuously long or short terminal branches or clades. mented in PAUP*4.0 (Swofford 2002) under the general time reversal plus gamma plus invariant sites (GTR-gþI) Quantile regression model. Because these ML distances could be biased if Conventional regression analyses describe changes in mitochondrial divergence is not constant across lineages, mean response, which might not be biologically mean- as would be the case if a subset of the taxa included here ingful information under some circumstances, including had a higher or lower mutation rate than the remaining our examination of the relationships between phyloge- taxa, we also calculated pairwise distances using the netic and ecological distances. While the potential for summed branch lengths connecting each pair of termini high ecological overlap could change with greater in an ultrametric tree. We derived this ultrametric tree phylogenetic distance between warbler species, this from an analysis in which the Bayesian topology potential might not be realized for all pairs of species. described below was imported into PAUP* as a Thus, on average, ecological overlap could vary little constraint tree, the heuristic search algorithm was set with changes in phylogenetic distance, even if some to produce a clocklike (ultrametric) topology, and a proportion of species pairs that are more distantly maximum-likelihood analysis was conducted using related do have greater ecological overlap; i.e., a mean GTR-gþI parameters derived from the Bayesian regression through the mean could show no relationship Markov chain Monte Carlo (MCMC) analysis. Here- between phylogenetic distance and ecological overlap, after, we use the term ‘‘ML distances’’ to refer to the but a regression through data from the most highly nonultrametric distance matrix, and ‘‘ultrametric dis- overlapping species (e.g., the 95th percentile of highest tances’’ to refer to the ML distance matrix derived from overlap at each value of phylogenetic distance) would the ultrametric branch lengths in the topology with an exhibit a relationship between phylogenetic distance and enforced molecular clock. For all statistical analyses ecological overlap. Thus, we needed to analyze our data involving genetic distance matrices, we report the results using statistical techniques that could provide us from the ML distances followed by the ultrametric regression lines through percentiles (quantiles) of our distances (hence these summaries each have two choosing, if we wanted to detect the patterns that we successive P values, etc.). As the results from the two expected. S20 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Ecology Special Issue

We did not simply select the data points from the Randomizations and statistical analyses quantiles of our choosing and run conventional regres- Two attributes of the data require the use of sion lines through these. This approach would have randomization tests for exploring the association required us to arbitrarily divide the continuous variation between ecological co-occurrence and phylogenetic in phylogenetic distance into discrete intervals, in which distance. First, the underlying comparisons between to determine the appropriate value of co-occurrence for pairs of species are not independent: owing to differ- each quantile used. Such arbitrariness would have meant ences in abundance, each pair of species was represented that regression lines, and thus biological conclusions, twice in calculations of ecological co-occurrence (A with would be dependent on the rules used to divide data into B, and B with A), and a given species was also rep- groups with differing ranges of phylogenetic distance. In resented in all pairwise associations with co-occurring order to avoid these complications in interpretation of species. Each species was similarly represented multiple results, we used a statistically justifiable and repeatable times in the matrix of pairwise genetic distances. As a analytical technique, linear quantile regression (Koenker result, a nonzero slope for the relationship between and Bassett 1978, Cade and Noon 2003), to quantify the phylogenetic distance and co-occurrence could be gen- relationship between phylogenetic distance and quan- erated by chance alone. This nonindependence would tiles of the distribution of co-occurrence. Instead of also cause true Type I error rates to differ from the describing a single line through the mean, as is done in nominal rates calculated by more conventional statis- conventional linear regression, a linear quantile regres- tical tests, another problem that is dealt with by sion describes linear changes in the shape of the entire randomization tests. distribution of the response variable at all values of a For our examination of local (within-BBS-transect) predictor variable, allowing a user to request production patterns, we generated null distributions against which of regression lines through one or several arbitrarily to compare the observed relationships between phylo- chosen quantiles of the response variable’s distribution. genetic and ecological co-occurrence by randomly Regression equations for any arbitrary quantiles of this shuffling species identities among points within each distribution can be calculated. BBS transect. The algorithm used in the randomizations In our analyses, we examined changes in median and was to randomly reorder the values within the species 5% quantiles of our co-occurrence measures as a name column in the input data table, with the reordering function of changes in phylogenetic distance. Remember done separately and independently for data from each that our indices of co-occurrence were defined so that BBS transect. The input data table for this random- high numbers correspond to lower co-occurrence; thus a ization had one line for each species and census point on 5% quantile denotes the 95th percentile of high co- a BBS transect on which that species was observed, with occurrence. Regressions through the median inform us each BBS transect being represented by only one year of data. The result of the randomization was to maintain as to whether pervasive changes in co-occurrence were the same number of warbler species being recorded at seen along the gradient of phylogenetic distances. A each point on a transect, maintain the number of points median-quantile regression line and a conventional at which each species was reported, and maintain the linear regression line would be essentially identical for same species composition on a transect as was present in data with normally distributed errors. Slopes from the the actual data. The randomization procedure was 5% quantiles, in contrast, indicate whether the extent of designed to break up local ecological associations co-occurrence varied only for a small proportion of between species, but still (1) maintain the identities species pairs with greater than median co-occurrence. and relative abundances of all species within each The 5% quantile reflects variation in the relationship transect and (2) maintain information on the proportion between co-occurrence and phylogenetic distance when of points within a transect that were suitable for any only a small proportion of species pairs have actually species of wood-warbler, for example, ensuring that realized the potential for greater co-occurrence with points with no reported warblers continued to have no changing phylogenetic distance. Note that, as mentioned warbler species after the randomization. This random- above, changes in the 5% quantile reflect changes in the ization procedure is similar to a Mantel’s test in which shape of the entire distribution of co-occurrence, and cells in a matrix of similarity values are randomly should not be interpreted to mean that only the 5% of rearranged to produce null distributions. Our procedure most highly co-occurring species varied in co-occurrence differed from that in a Mantel’s test by (1) constraining with changes in phylogenetic distance. We do not the randomization as explained and (2) employing a present results for 95% quantiles, as the 95% quantile different test statistic, in our case slopes from quantile of co-occurrence did not vary with changes in phyloge- regression. netic distance. In all cases the 5% of species pairs with In our analyses of among-transect associations the lowest co-occurrence (i.e., the 95% quantile) abutted between phylogenetic and regional co-occurrence, we the hard boundary of complete non-co-occurrence at used a similar randomization procedure. Both the point or transect (i.e., had a co-occurrence of zero and number of warbler species per transect and the total hence an ecological distance of 1.0). number of times each species was found in the data table July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S21 remained identical to the values in the real data. Only We calculated probabilities from our regressions of the associations between species pairs were broken up by the relationships between phylogeny and ecological the randomization procedure. Mechanistically, the overlap as the proportion of randomizations in which randomization was done on an input data table that the regression slope from the true data was greater than contained a column identifying each transect uniquely, the slope from the randomized data. Counts of steeper and another column containing species’ names. Each positive and negative slopes were tallied separately, and transect appeared 43 times in the file, one time for each the proportion of steeper slopes in one direction gave a warbler’s name. A third column in the input data was a one-tailed probability. The two-tailed probabilities that Boolean variable that indicated whether each species in we report in the text were calculated by doubling these the real data was reported on that specific transect. Each one-tailed probabilities. The confidence intervals de- transect was represented by a single year’s data in the picted in the figures are the range of the central 95% of input data file. The randomization was accomplished by all regressions. Plotted results from quantile regression randomly reordering the warbler species’ names relative analyses are not derived from median regression to the rest of the information in the input file; this coefficients, but are lines that connect a series of reordering was done separately for species that were predicted values, with the median predicted values listed as having been seen, and those listed as having not representing the regression line and the range of the been seen on a specific transect in the real data. These central 95% of predicted values representing the 95% two separate reorderings were needed to keep the confidence limits at each point. As a result of plotting number of transects on which a species was present in lines connecting point estimates, it is possible for plotted the randomized data at the same value as in the real lines to show deviations from perfect linearity even data. Although the transect-level results are interesting though linear quantile regressions were used. in comparison to the within-transect results that include We used the SAS statistical software to generate only species pairs with demonstrated regional sympatry, random data sets for analysis and the quantreg library it is important to note that the transect-level random- (Quantreg package, 2004, version 3.35, by R. Koenker ization brings together species that never occur together [available online])2 for the R statistical software (R in nature, because they have nonoverlapping geographic Development Core Team 2004) to conduct all quantile distributions. regression analyses. Conclusions did not change qual- The second reason for using randomization tests was itatively between analyses based on 500 and 1000 to incorporate replication across multiple years for each randomization iterations, indicating that 1000 iterations transect, as occurrences may differ among years for were sufficient to describe variability in null associations biological reasons (e.g., demographic productivity in the among taxa. previous year) and as sampling artifacts. We accounted for this interannual variation by generating multiple RESULTS AND DISCUSSION data sets, each of which contained a single, randomly Patterns of association within local areas (BBS transects) selected year’s data for each transect. We combined the Three features are immediately obvious in compar- two levels of randomization by (1) randomly selecting isons of ecological dissimilarity with phylogenetic one year for each transect and (2) creating a null data set distance (Fig. 2a). First, although pairwise genetic by randomizing species’ distributions for that transect- distances varied within 1–18%, the majority of pairwise year as we have described. This two-stage random- distances were clustered at intermediate levels of ization was repeated 1000 times each for the within- divergence owing to several periods of rapid clado- transect analyses and the among-transect analyses, and genesis during the diversification of this group (Lovette we separately performed the quantile regression analyses and Bermingham 1999, 2002). This temporal clustering for each iteration on the true and randomized data. of nodes is visible in the phylogenetic reconstruction for We investigated whether the patterns found in our these taxa (Fig. 3). Second, there is a notable lack of analyses were substantially influenced by single species taxa that are both phylogenetically similar and have through analyses that tested individually for the high local co-occurrence. This is seen in the empty lower influence of each species on the overall results. In these left quadrant in Fig. 2a. Third, across the full gradient of influence analyses, all species pairs containing data from genetic divergence, most species pairs detected on the a target species were removed from the data set, and same Breeding Bird Survey (BBS) transect were rarely quantile regressions were calculated for each of the 1000 detected at the same transect points, leading to the randomly sampled years of true data, as just described. majority of pairwise comparisons of ecological dissim- We used the differences between regression slopes with ilarity being clustered near the upper boundary in Fig. and without the target species as our metric of influence 2a. A smaller number of species pairs co-occur at higher of this species on the overall results. We treated each of frequency, with a median (of 1000 random subsamples the 43 species in turn as the target species and compared of BBS data) of only 0.085% of the 1184 pairwise their influences by examining median changes in regression slopes, from the 1000 data sets, when that species’ data were excluded. 2 hhttp://cran.r-project.org/doc/packages/quantreg.pdfi S22 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Ecology Special Issue

FIG. 2. Ecological co-occurrence of wood-warbler species pairs across a gradient of phylogenetic divergence (maximum- likelihood distances). In each panel, the background of plotted points depicts the same random sample of real census transect-years that is representative of patterns observed across all 1000 such iterations. (a) Distribution of pairwise associations between phylogeny and ecological co-occurrence among taxa that forage at similar (green) or different (black) habitat strata. (b) Median (thick lines) and lower fifth quantile (thin lines) relationships between phylogenetic distance and ecological co-occurrence for species pairs that forage in different habitat strata. Regression through the true data (red) and randomized null-model data (blue) are both plotted. For each of the four lines, the median from the 1000 iterations is plotted with the inner 95% of predicted values forming the confidence intervals (dashed lines). (c) Median and lower fifth quantile regressions for species pairs foraging in the same habitat stratum. (d) Corresponding quantile regressions for pairs of taxa foraging in different strata, but with data from Ovenbirds (Seiurus aurocapilla) excluded from the analyses. In panels (b) and (d), regression lines are only plotted across the range on the x- axis of actual data points used to derive the regressions. In these panels, the background points represent the relevant subset of data from the full distribution in panel (a). comparisons involving taxa with .50% detected co- local co-occurrence, although across all phylogenetic occurrence. On a historical note, the five ‘‘spruce-woods distances the majority of species pairs had relatively high warblers’’ featured in Robert MacArthur’s 1958 study local ecological dissimilarity. We contrasted these all have median (of 1000 random subsamples of BBS observed patterns with a null model of randomized data) pairwise dissimilarity values .0.83, suggesting species occurrences. When all pairs of taxa are that these species are not usually detected in local considered, the median co-occurrence does not change sympatry. significantly with phylogenetic distance relative to the Our interpretation of the conspicuous absence of null distribution (P ¼ 0.192 for differences in slopes species pairs in the lower left quadrant of Fig. 2a is that compared to the null model, based on maximum- closely related taxa effectively exclude each other at a likelihood distances; P ¼ 0.164, based on ultrametric local scale, even when they occur in geographic distances). However, maximal co-occurrence sig- proximity. Some wood-warbler species pairs separated nificantly increases as ML distance increases (P ¼ 0.03 by greater phylogenetic distances had high likelihoods of from the fifth-quantile regression slope), although the July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S23

FIG 3. Phylogenetic relationships among the 43 wood-warbler taxa included in this study based on Bayesian Markov chain Monte Carlo analysis of long mitochondrial DNA sequences (see Methods). Thick branches indicate species (or groups of species) coded as terrestrial foragers; the remaining taxa are arboreal foragers. The tree is rooted to various non-wood-warbler outgroup taxa, as in Lovette and Bermingham (2002). pattern only approached statistical significance when pairs (P ¼ 0.026 for ML distances, P ¼ 0.000, for ultra- ultrametric distances were used (P ¼ 0.066). These metric distances), for which fifth-quantile co-occurrence results suggest that a subset of the pairs of more increased with greater phylogenetic distance (Fig. 2b), distantly related species is capable of higher local but not for same-stratum pairs, for which the slopes of association than expected by chance. the real and randomized regressions do not differ Further analyses suggest that the increased co- significantly (P ¼ 0.526 for ML distances, P ¼ 0.736 for occurrence of some more distantly related species pairs ultrametric distances) (Fig. 2c). Results from analyses requires divergence in foraging niche in order to reduce using ultrametric distances even suggested the possibil- potential competition. Adding each species pair’s forag- ity of higher co-occurrrence with greater phylogenetic ing strata information (in the form of a categorical distance from the median regression for mixed-substrate variable with two categories) to the quantile regression pairs (P ¼ 0.070), although this pattern was not analyses revealed a nonrandom relationship at the reflected in the median regression when ML distances lower (fifth-quantile) boundary for the mixed-strata were used (P ¼ 0.236). No hint of nonrandom S24 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Ecology Special Issue

associations were found from the median regressions models, indicates that, regardless of phylogenetic from same-substrate pairs (P ¼ 0.556 for ML distances, distance, similarly related sympatric species choose a P ¼ 0.478 for ultrametric distances). Thus, in spite of larger number of similar habitats than expected by already having a greater average genetic distance chance. This pattern is consistent with a growing body associated with their differing foraging substrates of evidence showing that phylogenetic relationships (compare the ranges of genetic distances in Fig. 2b, sometimes explain substantial variation in how related c), only the mixed-stata pairs showed changes in co- species are distributed across habitat types, bioclimatic occurrence with phylogenetic distance. This result regions, and other niche-related environmental variables suggests that differences in or associated with foraging (e.g., Ricklefs and Latham 1992, Peterson et al. 1999). substrate are a necessary but not sufficient precondition The historical legacy in ecological niches is not surpris- for co-occurrence to vary with phylogenetic distance. ing, given that phylogenetic effects are usually apparent To explore which taxa were driving the fifth-quantile in organismal traits with a genetic basis, from molecular relationship between phylogeny and ecological overlap pathways to complex behaviors (Harvey and Pagel 1991, (Fig. 2b), we repeated these analyses and serially Ackerly 2003). In a different context, niche conservation removed comparisons involving each of the 43 taxa. is also apparent in the distribution of arboreal vs. These influence analyses were only conducted on the terrestrial foraging among these 43 wood-warbler fifth-quantile regressions for mixed-strata pairs, as this species (Fig. 3): evolutionary switches among these was the only case in which a clear effect of phylogenetic strata are uncommon, and no pairs of very closely distance on ecological co-occurrence was detected. related species have diverged across this level of spatial Median changes in the fifth-quantile slope, with removal segregation. of data from a single species, almost all clustered between 0.5 and 0.5 for the analyses based on ML Are patterns driven by differences in species’ detection probabilities? phylogenetic distances, and between 0.2 and 0.08 for analyses based on ultrametric distances. The only The data presented in Fig. 2 are based on detections exceptions were for Ovenbirds, in whose absence the of individual birds, which require both that a given median increase in slope was 2.9 (2.7 with ultrametric species is actually present and also that this species has distances) (Fig. 2d) and Palm Warblers (Dendroica been detected by the censusing observers. Actual palmarum), for which the median change in slope was presence, and hence ecological co-occurrence, is thereby 1.6 1.2 for ultrametric distances). Only the increase in systematically underestimated by the inevitable missed regression slope in the absence of Ovenbirds is relevant detections. As a result, the percentages of co-occurrence to explaining the increased ecological association with presented here are not direct quantifications of increasing phylogenetic distance, and for analyses based ecological co-occurrence, but instead comparable in- on ML phylogenetic distances only for Ovenbirds was dices of relative co-occurrence. Nevertheless, examina- the inner 95% of changes above zero, with none of the tion of patterns in our index of detection probability 1000 data sets showing a lower slope when Ovenbirds (see Methods) leads us to believe that the general were excluded from the quantile regressions. With patterns of ecological co-occurrence that we have ultrametric phylogenetic distances, the slope of the 5% summarized would be essentially impossible to create quantile line was always higher in the absence of data as a result of among-species variation in detection from Ovenbirds and always lower in the absence of data probability alone. from Palm Warblers. The effect of Ovenbirds is not We expect that relatively low ecological co-occurrence surprising because of the Ovenbird’s placement as the for the majority of species pairs is a biologically real earliest branch within the entire Parulidae (Lovette and phenomenon, because creation of this pattern through Bermingham 2002; I. J. Lovette, unpublished data) variation in detection probability would require at least (Fig. 3). If ecological differentiation is associated with one member of the majority of species pairs to have an time, this relatively ancient lineage is expected to exhibit anomalously low detection probability. As long as the the highest ecological differentiation (and concomitant distribution of species’ detection probabilities has a spatial association) relative to the more derived species mode at some intermediate value (as our index of within the radiation. detection probability does; see Methods), the observed A nonrandom increase in the potential for ecological distribution of ecological co-occurrence would be co-occurrence of distantly related species was one of two extremely unlikely to be produced as a result of patterns evident in our data. We also find evidence of interspecific variation in detection rates. The products consistent phylogenetic niche conservatism in the of detection indices for all observed species pairs are, as ubiquitous vertical offset between the actual and null expected, distributed with a peak at intermediate values, quantile regression lines (Fig. 2b–d). This phenomenon which indicates that the generally low local co-occur- is present in all quantile regressions and is particularly rence of species pairs (Fig. 2a) is a biologically real evident in the lower fifth quantiles. The consistently phenomenon. lower position of the regression lines based on the real For reasons similar to those we have discussed, in- data, relative to the lines from the corresponding null creasing ecological co-occurrence with increasing phy- July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S25

FIG. 4. Regional co-occurrence of wood-warbler species pairs across a gradient of phylogenetic divergence (maximum- likelihood distances). In each panel the background of plotted points depicts the same random sample of real census transect-years that is representative of patterns observed across all 1000 such iterations. (a) Distribution of pairwise associations between phylogeny and regional co-occurrence among taxa that forage at similar (green) or different (black) habitat strata. (b) Median (thick lines) and lower fifth quantile (thin lines) relationships between phylogenetic distance and regional co-occurrence for all species pairs combined. Regressions through the true data (red) and randomized null-model data (blue) are both plotted. For each of the four lines, the median from the 1000 iterations is plotted with the inner 95% of predicted values forming the confidence intervals (dashed lines). logenetic distance (Fig. 2b) also appears highly unlikely only 8% across the full range of phylogenetic distances to be an artifact of systematic variation in detection between Ovenbirds and arboreally foraging warblers probabilities among species as a function of their found on the same transects. In contrast, the presence of phylogenetic distance. For variation in detection prob- species pairs with Ovenbirds leads to a roughly 135% abilities to have caused this pattern, we would need to change in ecological co-occurrence at the fifth quantile find detection probabilities of some proportion of (Fig. 2d). This contrast of effects suggests that, while species pairs to increase with increasing phylogenetic systematic variation in detectability contributed slightly distance. However, we found no evidence for this, and in to the closer measured ecological co-occurrence of fact a significant tendency in the opposite direction using arboreally foraging warblers more distantly related to a fifth-quantile regression of minimum detection index Ovenbirds, the effect of this measurement artifact was (within each of the 903 species pairs) against phyloge- trivial. netic distance. In this fifth-quantile regression, con- Finally, the lower position of the real vs. null fidence intervals around the estimated slope did not regression lines that we interpret as indicating higher come close to overlapping zero (slope ¼0.45, 95% than expected niche conservatism could only be an confidence limits of 0.50 to 0.41). Thus, we believe artifact of varying detection probabilities if the actual that the increasing ecological co-occurrence with greater species pairs were composed of pairings in which both phylogenetic distance is a biological phenomenon and members had higher detection probabilities than was not an artifact of limitations of data collection. typical of randomly selected pairs of warbler species. Interspecific variation in detection rates could only The median product of the detection probabilities of the have caused the significant influence of Ovenbirds on the actual species pairs was 0.281, and products ranged results (Fig. 2b, d) if increasing genetic distance from from 0.198 to 0.375 in the 903 observed species pairs. Ovenbirds were correspondingly correlated with increas- From 1000 iterations of an equal number of random ing detection probability (e.g., by louder and more species pairings, the median product was 0.281 (95% persistent vocalization) for arboreally foraging wood- confidence limits 0.279–0.282). There is no evidence to warblers. While there was an increase in detection suggest that phylogenetic niche conservatism is a probability of arboreally foraging species paired with sampling artifact. Ovenbirds as their phylogenetic distance from Oven- birds increased, the result was not statistically significant Contrasting local and regional relationships between (r2 ¼ 0.05, P ¼ 0.19, N ¼ 33, slope ¼ 0.76). Even were this phylogenetic distance and spatial co-occurrence increase in detection probability real, it still would At the broader spatial scale involving comparisons translate to an average change in the detection index of among transects, there was a greater range of variation S26 IRBY J. LOVETTE AND WESLEY M. HOCHACHKA Ecology Special Issue

in species co-occurrence (Fig. 4a) than was found for (e.g., Barraclough and Vogler 2000), but the lack of such local, ecological co-occurrence (Fig. 2a). However, the an association in our analyses is the result predicted for higher values of co-occurrence may largely be accounted groups of organisms—such as these warblers—in which for by the higher probability of detection of a given species’ ranges must have been shifted repeatedly by species on a transect as a whole, relative to a single point large-scale climate cycles, thereby scrambling most on that transect. Nevertheless, we found a predominance geographic patterns associated with their initial speci- of species pairs with little or no overlap in occurrence on ation events (Losos and Glor 2003). transects, as we would expect given that at maximum 23 In summary, when comparing point (Fig. 2) and of the 46 North American wood-warblers ever occur on transect (Fig. 4) scales, there are clear differences in both the same transect, and most transects support substan- the general magnitude of species’ co-occurrence, and in tially fewer species (Fig. 1). This real geographical the relationship between phylogenetic distance and separation among many species pairs drives much of the species co-occurrence. Broadly speaking, these differ- pattern in the transect-level analyses and demonstrates ences reinforce the notion that the factors that are the value of the point-based comparisons we describe, in determining local ecological co-occurrence are different which we are able to restrict analyses to only those than those biogeographic influences that have created transects where both members of a given pair of species the varying distributional ranges of North America’s occur. parulid warblers. At the transect level, the fact that many pairs of warbler species have largely to fully nonoverlapping CONCLUSIONS ranges is evidenced in the y-axis offsets between actual Phylogenetic niche conservation has most often been median regional overlap and the overlap expected by observed in allopatric species that occupy similar niches chance (Fig. 4b). Here, the median regional overlap was despite a period of evolutionary isolation (e.g., Peterson lower (rather than higher, as in the point-based analyses) et al. 1999); in such cases, niche conservation may even than expected by chance, as indicated by the complete enhance the probability of continued isolation if it displacement of the median-quantile regression line lowers the likelihood that one or both populations above the null-model regression line. The opposite evolve ecologically to occupy the previously unsuitable pattern was found in the fifth-quantile regressions, for habitat that separated them (Wiens 2004). Stabilizing which the observed level of regional co-occurrence was selection on niche attributes is one potential cause of higher, for the 5% of most overlapping species, than that phylogenetic niche conservation (Harvey and Pagel expected by chance alone, regardless of phylogenetic 1991, Ackerly 2003), but stabilizing selection leading to distances. This fifth-quantile result is likely a function of phylogenetic niche conservatism may be more likely in some species pairs having very similar habitat associa- allopatry because it is less likely to be opposed by tions, and thus ranges. divergent selection among closely related sympatric The final pattern to emerge from analyses of regional taxa. The analyses here suggest that niche conservation association is that variation in regional overlap with is evident in a large group of species, even with phylogenetic distance is different from that expected by substantial sympatry. In the wood-warblers, divergent chance alone, when analyses were based on ML selection stemming from interspecific competition has distances. If warbler species were randomly distributed not been sufficient to completely erase the phylogenetic across North America, the randomization suggests that legacy of habitat specialization. there would be a slight decrease in regional co- The majority of our comparisons address only the occurrence with increasing phylogenetic distance (as component of ecological differentiation that is associ- indicated by the positive slope of the blue median- ated with species’ spatial co-occurrences, or their lack quantile regression line in Fig. 4b). What we actually thereof. For example, these comparisons are much more found for the real data is no relationship between likely to detect patterns related to fundamental differ- regional co-occurrence and phylogenetic distance (as ences in species’ selection of habitats than they are to the represented by the flat red median-quantile in Fig. 4b), a more subtle ecological differences that likely allow co- statistically significant difference from the null slope (P existing species to partition ecological resources within ¼ 0.000). For analyses based on ultrametric distances, habitats. The contrasting quantile regressions for pairs the slope of the line through the real data did not differ of warbler species that forage in similar vs. mixed from zero, although in this case the result was not a vegetation strata (Fig. 2b, c) demonstrate that behav- departure from the null regression line (P ¼ 0.110), the ioral differentiation contributes to patterns of local slope of which also did not differ from zero. This community composition, but we were unable to further comparison suggests that there is only a weak signature address this level of ecological niche differentiation. of phylogeny on the overall geographic ranges of these Another important limitation of our approach is that North American warbler species. Previous studies of both present and past competition might cause species other taxa have sometimes reported stronger (and to be spatially segregated, and our analyses do not usually positive) correlations between phylogenetic distinguish between the effects of ongoing species distances and the overlap of species’ geographic ranges interactions and differentiation in site selection caused July 2006 WARBLER PHYLOGENY AND COMMUNITY STRUCTURE S27 by competition in the past. Similarly, at an even broader Anderson, T. M., M. A. Lachance, and W. T. Starmer. 2004. spatial scale, it is possible that competitive interactions The relationship of phylogeny to community structure: the cactus yeast community. American Naturalist 164:709–721. feed back to influence species’ ranges, particularly if Barraclough, T. G., and A. P. Vogler. 2000. Detecting the species with highly similar ecological niches tend to geographical pattern of speciation from species-level phylog- exclude one another at this broad scale. The influence of enies. American Naturalist 155:419–434. competition on the regional species pool has been Cade, B. S., and B. R. Noon. 2003. A gentle introduction to termed the Narcissus Effect in the ecological literature quantile regression for ecologists. Frontiers in Ecology and the Environment 1:412–420. (Colwell and Winkler 1984), but has received less Cavender-Bares, J., D. D. Ackerly, D. A. Baum, and F. A. attention in a phylogenetic context. Because the Bazzaz. 2004. 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APPENDIX Alternative versions of Figs. 2 and 4, based on ultrametric genetic distances (Ecological Archives E087-109-A1). Ecology, 87(7) Supplement, 2006, pp. S29–S38 Ó 2006 by the Ecological Society of America

PHYLOGENETIC ANALYSIS OF THE EVOLUTION OF THE NICHE IN LIZARDS OF THE ANOLIS SAGREI GROUP

1,4 2 3 2 JASON H. KNOUFT, JONATHAN B. LOSOS, RICHARD E. GLOR, AND JASON J. KOLBE 1Department of Ecology and Evolutionary Biology and University of Colorado Museum, UCB 265, University of Colorado, Boulder, Colorado 80309 USA 2Department of Biology, Campus Box 1137, Washington University in St. Louis, St. Louis, Missouri 63130 USA 3Center for Population Biology, University of California, Davis, California 95616 USA

Abstract. Recent advances in ecological niche modeling (ENM) algorithms, in conjunction with increasing availability of geographic information system (GIS) data, allow species’ niches to be predicted over broad geographic areas using environmental characteristics associated with point localities for a given species. Consequently, the examination of how niches evolve is now possible using a regionally inclusive multivariate approach to characterize the environmental requirements of a species. Initial work that uses this approach has suggested that niche evolution is characterized by conservatism: the more closely related species are, the more similar are their niches. We applied a phylogenetic approach to examine niche evolution during the radiation of Cuban trunk-ground anoles (Anolis sagrei group), which has produced 15 species in Cuba. We modeled the niche of 11 species within this group using the WhyWhere ENM algorithm and examined the evolution of the niche using a phylogeny based on ;1500 base pairs of mitochondrial DNA. No general relationship exists between phylogenetic similarity and niche similarity. Examination of species pairs indicates some examples in which closely related species display niche conservatism and some in which they exhibit highly divergent niches. In addition, some distantly related species exhibit signiﬁcant niche similarity. Comparisons also revealed a specialist–generalist sister species pair in which the niche of one species is nested within, and much narrower than, the niche of another closely related species. Key words: anole; Anolis; Cuba; ecological niche modeling; fundamental niche; niche conservatism; niche evolution; phylogenetics.

INTRODUCTION global climate and land cover Geographic Information The fundamental niche, which encompasses the System (GIS) and remote-sensing data has provided theoretical range of conditions a species can occupy environmental information at a regional scale. These (Hutchinson 1957), provides a conceptual framework to data, when integrated into ecological niche modeling predict the potential geographic distribution of a species (ENM) algorithms, have provided a powerful oppor- (MacArthur 1972, Sobero´n and Peterson 2005). Species tunity to characterize the habitat requirements of a traits, whether morphological, physiological, or behav- variety of species and assess patterns of niche differ- ioral, are often obviously linked to the niche and entiation in a comparative framework. The majority of generally susceptible to the processes of evolution. recent niche modeling efforts have focused on predicting Consequently, the shaping of niche characteristics can species’ distributions, species’ response to climate be viewed as an evolutionary phenomenon. Because the change, and potential distributions of invasive species fundamental niche provides details about the potential (Guisan and Zimmerman 2000, Peterson 2001, 2003, distribution of species, and the niche is determined by Peterson and Vieglais 2001, Oberhauser and Peterson the processes of evolution, understanding evolutionary 2003, Peterson and Robins 2003, Illoldi-Rangel et al. patterns of niche diversiﬁcation can reveal valuable 2004, Peterson et al. 2004). While these studies have insights into factors related to the diversity and provided novel insights into aspects of broad-scale distribution of species. ecological niche characteristics, recent research has Studies of the niche have historically involved detailed begun to realize the potential to examine the results of analyses of local habitat requirements of an organism niche modeling efforts in an evolutionary context (Chase and Leibold 2003). Recently, the availability of (Peterson et al. 1999, Rice et al. 2003, Graham et al. 2004). Two approaches have been taken to integrating Manuscript received 25 January 2005; revised 16 August information on evolutionary relationships into niche 2005; accepted 24 August 2005. Corresponding Editor (ad hoc): modeling studies. On one hand, some studies have C. O. Webb. For reprints of this Special Issue, see footnote 1, p. S1. focused on recent evolutionary events by comparing 4 E-mail: [email protected] pairs of closely related taxa (either subspecies or sister S29 S30 JASON H. KNOUFT ET AL. Ecology Special Issue

avoiding the pitfalls of ancestor reconstruction. Specif- ically, following the logic of Harvey and Pagel’s (1991) ‘‘non-directional approach,’’ we examine the extent to which niche similarity among extant species is a function of phylogenetic similarity. Like the widely used independent-contrasts method (Felsenstein 1985), this approach allows the integration of phylogenetic information into comparative analyses without requiring inference concerning the characteristics of hypothetical ancestral taxa. Caribbean lizards of the genus Anolis are a particularly good group for such studies (see Plate 1). Anoles are abundant and diverse on Caribbean islands, with as many as 57 species on a single island (Cuba) and up to 11 species occurring sympatrically, and their ecology has been extensively studied (reviewed in Losos [1994] and Roughgarden [1995]). Moreover, recent research has established a firm phylogenetic framework for the Caribbean anole radiation (Poe 2004, Nicholson et al. 2005). Our focus in this study is on the A. sagrei species group on Cuba. This clade contains 15 species, all but one of which use similar microhabitats, occurring on tree trunks and other broad surfaces low to the ground, FIG. 1. Ultrametric phylogeny for the Anolis sagrei group and using the ground extensively for foraging and derived from Bayesian phylogenetic analysis of ;1500 base intraspecific interactions (the ‘‘trunk ground ecomorph’’ pairs of mtDNA. The support values and simplified tree presented here were generated by culling taxa from a larger tree niche of Williams [1983]). Several species occur widely that included 306 unique sequences obtained from 315 throughout Cuba, whereas others have restricted dis- individuals representing 11 of 15 A. sagrei group species. tributions; as many as four occur sympatrically (Losos Numbers above nodes represent posterior probabilities ob- et al. 2003). Recent phylogenetic work (Glor 2004) has tained from Bayesian analysis. Boldface numbers below nodes represent bootstrap values obtained from 200 bootstrapped provided the phylogenetic framework for the investiga- data sets analyzed via maximum parsimony. tion of the evolution of the niche and community composition. species [Peterson et al. 1999, Peterson and Holt 2003]). In this study, we examine the evolution of niche Results from these studies suggest that conservatism, characteristics in species within the A. sagrei group on where taxa that are more closely related possess Cuba. We characterize the broad-scale environmental characteristics that are more similar (Harvey and Pagel components of the niche using ENM algorithms and 1991, Lord et al. 1995, Webb et al. 2002), is a frequent GIS data. We then incorporate the results of these occurrence and characterizes evolutionary patterns of analyses with information on the phylogenetic relation- niche diversification. Alternatively, other studies have ships within the A. sagrei group to examine patterns of included deeper evolutionary divergence by using niche diversification within this island group. phylogenetic methods to reconstruct the niche of METHODS ancestral taxa (Rice et al. 2003, Graham et al. 2004). Results from these studies suggest that niche conserva- Phylogenetic relationships among members tism might not be a consistent characteristic during of the Anolis sagrei group diversification. Both of these approaches have limita- We obtained an mtDNA phylogeny for the A. sagrei tions: the former approach has limited scope and only species group from Glor (2004). This tree was recon- applies to evolutionary events in the recent past, whereas structed from 306 unique sequences obtained from 315 the accuracy of the latter approach is questionable individuals representing 11 of 16 A. sagrei group species because a number of studies have demonstrated that and including extensive intraspecific sampling within phylogenetically derived estimates of ancestral states will most widespread species (Glor 2004). We treat A. allogus in many cases have low accuracy and extremely high as two separate species, A. allogus (east) and A. allogus uncertainty (Schluter et al. 1997, Losos 1999, Martins (west), because they are highly divergent genetically and 1999, Oakley and Cunningham 2000, Webster and do not form a clade (Glor 2004) (Fig. 1); thus the Purvis 2002). discrepancy between the 15 recognized species and the We propose an alternative approach, similar to one 16 species referred to in our analyses. Maximum- applied by Rice et al. (2003), that makes full use of parsimony analysis implemented by PAUP* 4.0b10 phylogenetic information and thus permits inferences (Swofford 2002) and Bayesian analysis implemented in beyond comparison of closely related taxa, while MrBayes 3.0 (Huelsenbeck and Ronquist 2001) yielded July 2006 NICHE EVOLUTION IN ANOLIS LIZARDS S31

TABLE 1. Results of niche modeling predictions for species in the Anolis sagrei group.

WhyWhere GARP external accuracy Species N mean (range) External accuracy Z statistic A. ahli 16 0.748 (0.500–0.999) 0.490 31.15*** A. allogus (east) 65 0.542 (0.466–0.972) 0.758 23.19*** A. allogus (west) 23 0.500 (0.500–0.500) 0.945 29.26*** A. bremeri 25 0.545 (0.500–0.952) 0.845 30.40*** A. homolechis 146 0.962 (0.951–0.972) 0.595 18.50*** A. jubar 63 0.594 (0.500–0.984) 0.736 22.00*** A. mestrei 35 0.597 (0.500–0.993) 0.958 30.13*** A. ophiolepis 43 0.814 (0.500–0.973) 0.756 19.16*** A. quadriocellifer 24 0.735 (0.500–0.992) 0.981 30.73*** A. rubribarbus 21 0.547 (0.500–0.971) 0.971 30.23*** A. sagrei 124 0.953 (0.937–0.968) 0.627 17.80*** ***P , 0.001 for all of the WhyWhere predictions. Number of localities for each species. congruent and well-supported topologies (Fig. 1). for predicting whether a species will occur in a particular Phylogenetic analysis of a nuclear DNA fragment (the region, but is not insightful regarding microhabitat third intron of the rhodopsin-encoding gene) for a differences among co-occurring species. subset of the taxa included in the mtDNA phylogeny We compiled locality information for 11 species in the also yields a topology that is concordant with all of the A. sagrei group from natural history museum collection nodes presented in Fig. 1 (Glor 2004). records (American Museum of Natural History; Califor- We then converted the Bayesian mtDNA tree into an nia Academy of Sciences; Field Museum; Museum of ultrametric form using Sanderson’s (2002) penalized- Comparative Zoology, Harvard University; Museum of likelihood approach, as implemented by the program r8s Vertebrate Zoology, University of California-Berkeley; (Sanderson 2003). A smoothing value for the penalized- Smithsonian National Museum of Natural History; Uni- likelihood analysis was determined via cross-validation. versity of Kansas Natural History Museum) and pub- Following conversion of the tree into an ultrametric lished data (Schwartz and Henderson 1991, Rodrı´guez- format, taxa within monophyletic groups were pruned Schettino 1999) (Table 1). The five species for which until a single representative of each species remained phylogenetic data are not available are known from an (Fig. 1). In the case of A. allogus, we retained two extremely limited number of individuals or localities individuals representing the genetically distinct eastern (Rodrı´guez-Schettino 1999). and western populations. This pruned tree was then used We predicted the niche of each species in the A. sagrei to derive patristic distances among all pairwise taxo- group using the WhyWhere niche modeling program 5 nomic comparisons. (Stockwell 2006; software available online). The Why- Where algorithm (newly available in June 2004) affords Niche characteristics of members greater predictive ability and decreased computational of the Anolis sagrei group time compared to the commonly used GIS-based Ecological niche modeling provides the ability to Genetic Algorithm for Rule-Set Prediction (GARP) estimate the niche of a species based on known species ENM application (Stockwell and Peters 1999). To localities and environmental parameters characterized in achieve results, WhyWhere converts environmental data GIS data sets. This estimation is then used to predict the layers into multicolor images and applies a data-mining potential geographic distribution of a species based on approach to image processing methods to sort through the same GIS data (Peterson 2001). These predictions large amounts of data to determine the variables that are based on broad-scale environmental data and do not best predict species occurrences. Testing of the predic- account for microhabitat characteristics. Consequently, tive ability of each model is performed by calculating the microhabitat partitioning among sympatric anoles, accuracy of the model based on species presence data which has been an important component in the and randomly generated pseudo-absence points within a community structure and evolutionary diversification specified geographic region (similar to GARP) (Stock- of anoles (e.g., Williams 1983, Losos et al. 2003), is not well and Peters 1999), in this case Cuba. considered in the ENM algorithms. Similar broad-scale We used the WhyWhere ENM algorithm, 184 ENM analyses have suggested a high predictive ability terrestrial environmental data layers (see Stockwell [2006] for information on data layers), and georefer- for geographic distribution with the ENM algorithms enced species occurrence data to predict the niche of (Peterson 2001, 2003, Peterson and Vieglais 2001, each species in the A. sagrei group. These ENM Oberhauser and Peterson 2003, Peterson and Robins 2003, Illoldi-Rangel et al. 2004, Peterson et al. 2004). In other words, the broad-scale ENM approach is useful 5 hhttp://biodi.sdsc.edu/ww_home.htmli S32 JASON H. KNOUFT ET AL. Ecology Special Issue

FIG. 2. Potential distributions of species in the Anolis sagrei group based on WhyWhere and Genetic Algorithm for Rule-Set Prediction (GARP) ecological niche modeling predictions. Points represent locality data used in model development and testing for each species. Species designations are as follows: A, A. ahli;B,A. allogus (east); C, A. allogus (west); D, A. bremeri;E,A. homolechis;F,A. jubar;G,A. mestrei;H,A. ophiolepis;I,A. quadriocellifer;J,A. rubribarbus;K,A. sagrei. predictions reflect the potential geographic distribution qualitative assessment of the WhyWhere application. of each species on Cuba based on the ENM algorithms Both GARP and WhyWhere were developed by the and the GIS data used to construct each prediction. same person/research group, and both applications Each prediction was calculated based on a 0.18 calculate model accuracy in a similar manner (Stockwell resolution per grid cell, a 0.5 occurrence cut for each and Peters 1999, Stockwell 2006). Thus, the accuracy of prediction, and a 1.28 Z score termination condition. predictions generated by the two ENM algorithms are For each species, 50% of the locality data were used for qualitatively comparable. model training (internal accuracy), while 50% were held Using the Anolis locality data and the GARP ENM back for testing of model accuracy (external accuracy). application, we again predicted the niche of each species We determined accuracy of each prediction using the in the A. sagrei group. GIS data sets were at a 0.18 same protocol as GARP (Stockwell and Peters 1999). resolution and included layers describing topography Although WhyWhere has been demonstrated to (elevation, slope, aspect, flow accumulation, and flow generate predictions with higher accuracy than the direction) and climate (annual means of total, minimum, frequently used GARP ENM (Stockwell 2006), we also and maximum temperature, precipitation, solar radia- modeled the niche of each species using GARP for tion, vapor pressure, and wet days). We developed 100 July 2006 NICHE EVOLUTION IN ANOLIS LIZARDS S33

FIG. 2. Continued. niche models for each species based on locality data. The We investigated the relationship between niche GARP algorithm was run for 1000 iterations or until a similarity and phylogenetic relatedness in a geographic convergence limit of 0.1 was achieved for each species. context by examining the frequency that known local- During model development, 50% of the localities were ities from a particular species fall within the ENM used for model training, while 50% of the localities were predicted geographic distribution of another species held back to test model accuracy. Using the best-subset (similar to Peterson et al. [1999]). Using a binomial selection criteria (Anderson et al. 2003), we chose 20 probability distribution, we determined if the number of models that had an omission error of ,5% based on the times that the occurrence data points of one species localities used to test each model. From these 20 ‘‘best’’ overlapped the predicted distribution of another species was nonrandom. For the binomial probability calcu- models, we then selected the 10 models that exhibited an lation, the null expectation was that the percentage of intrinsic commission index closest to the mean intrinsic actual occurrence data points that fell within the commission index for the 20 models (Anderson et al. predicted range of the other species would correspond 2003; similar to Oberhauser and Peterson [2003]). We to the proportion of Cuba lying within the predicted then imported these models into GIS software (DIVA- distribution range. For each species pair, we conducted GIS [Hijmans et al. 2001]), identiﬁed the areas of reciprocal tests for each species. In sister species pairs, predicted occurrence that were present in all 10 models, greater than expected overlap by both species indicates and used this area as our prediction for each species. The niche conservatism, and less than expected overlap in use of areas that only occur in the predictions of all 10 both species indicates niche divergence. Also, in sister models is a conservative approach; however, this species pairs, greater than expected overlap by one methodological choice still resulted in predictions that species and less than expected overlap by the other could encompassed all of the locality data for each species. suggest a case of niche specialization. In more distantly S34 JASON H. KNOUFT ET AL. Ecology Special Issue

TABLE 2. Pairwise comparisons of percentage of locality points of one Anolis species that fall within the predicted range of a second Anolis species.

Species 2 Species 1 A. ahli A. allogus (east) A. allogus (west) A. bremeri A. homolechis A. jubar A. ahli A. allogus (east) 4.6, 0.0 A. allogus (west) 4.3, 93.8 8.7, 6.2 A. bremeri 0.0, 93.8 4.5, 10.8 100, 91.3 A. homolechis 6.5, 68.8 59.4, 95.4 47.8, 95.7 39.9, 91.3 A. jubar 0.0, 0.0 60.3, 69.2 6.3, 0.0 14.3, 12.0 50.8, 39.1 A. mestrei 0.0, 93.8 0.0, 0.0 97.1, 95.7 97.1, 100 94.3, 35.5 8.6, 0.0 A. ophiolepis 11.6, 93.8 41.9, 73.8 58.1, 82.6 44.2, 100 90.7, 62.3 30.2, 42.9 A. quadriocellifer 0.0, 0.0 54.2, 0.0 91.7, 4.3 91.7, 26.1 66.7, 5.8 37.5, 0.0 A. rubribarbus 25.0, 0.0 40.0, 56.9 0.0, 0.0 0.0, 0.0 35.0, 18.1 40.0, 12.7 A. sagrei 10.2, 25.0 39.8, 86.2 51.7, 95.7 44.9, 100 70.3, 76.1 30.5, 57.1 Notes, Percentages in boldface represent cases in which a smaller than expected number of locality points of one species fall within the predicted range of the second species. Percentages in italics represent cases in which a greater than expected number of locality points of one species fall within the predicted range of the second species. In each cell, the value on the left represents the percentage of locality points from Species 1 that fall in the predicted range of Species 2. The value on the right represents the percentage of locality points from Species 2 that fall in the predicted range of Species 1.

related species pairs, greater than expected overlap could at each species locality. All climatic data were log10- suggest either niche convergence or suggest that the transformed to standardize data for statistical analyses. species have both retained the ancestral condition (i.e., A principal-components analysis (PCA) was per- stasis). formed on the correlation matrix of transformed In addition to the geographic approach to niche environmental data to generate an environmental overlap using species locality data and the predictions envelope for each species. To generate the environ- generated by the ENM algorithms, we also examined mental envelope, the ﬁrst two axes of the PCA for each overlap of the ‘‘environmental envelopes’’ of species species were plotted in x–y space using ArcGIS (version pairs using GIS-derived environmental data extracted 9.0). A minimum convex polygon (MCP) was then from localities for each species. We generated the calculated around the points for each species using the environmental envelope for each species based on data Hawth’s Tools extension in ArcGIS (available online).7 extracted from WorldClim Global Climate GIS data sets The area for each species MCP was then calculated in (30-second resolution; WorldClim interpolated global ArcGIS. The percentage niche overlap for each species terrestrial climate surfaces, version 1.3; data and pair was calculated as the ratio of the sum of the overlap software available online)6 (Hijmans et al. 2004). The areas in each species’ MCP to the total area of each WorldClim data sets consisted of 19 bioclimatic species’ MCP, with the resulting quotient multiplied by variables including annual mean temperature, mean 100 to yield a percentage. diurnal temperature range, isothermality, temperature While the geographic approach applied to the ENM seasonality, maximum temperature of warmest month, predictions and species locality data is useful for minimum temperature of coldest month, temperature assessing similarities between individual species pairs, annual range, mean temperature of wettest quarter, the environmental-envelope method allows for the mean temperature of driest quarter, mean temperature examination of the relationship between niche similarity of warmest quarter, mean temperature of coldest and phylogenetic similarity among all members of the quarter, annual precipitation, precipitation of wettest clade. The environmental envelope could not be used to month, precipitation of driest month, precipitation assess differences between individual species pairs, seasonality, precipitation of wettest quarter, precipita- because we do not know the breadth of the environ- tion of driest quarter, precipitation of warmest quarter, mental envelope for all of Cuba. Consequently, we could and precipitation of coldest quarter, with all temper- not apply a binomial probability calculation to the atures reported in degrees Celsius and all precipitation overlap between species pairs. We calculated the amounts reported in millimeters. Environmental data correlation between niche similarity (i.e., percentage for each species was compiled by importing species envelope overlap) and phylogenetic similarity (patristic locality points into DIVA-GIS (Hijmans et al. 2001) to distance between taxa) among all species pairs using a generate longitude–latitude layers for each species. Mantel test. A signiﬁcant negative correlation indicates Environmental data at each locality were then extracted niche conservatism between closely related species and from each GIS data set to provide 19 climatic measures niche divergence between distantly related species.

6 hhttp://biogeo.berkeley.edui 7 hhttp://www.spatialecology.com/htoolsi July 2006 NICHE EVOLUTION IN ANOLIS LIZARDS S35

TABLE 2. Extended. little overlap, and most of the cases of the greatest overlap are among distantly related species pairs.

DISCUSSION A. mestrei A. ophiolepis A. quadriocellifer A. rubribarbus A common finding related to trait evolution is that conservatism is the expected pattern during species diversification (Webb et al. 2002). In terms of niche evolution, this conservatism has been hypothesized to result from active, stabilizing selection (Lord et al. 1995), or from fixation of ancestral traits that limit the 48.8, 82.9 potential range of outcomes during niche evolution 91.7, 11.4 41.7, 7.0 (Westoby et al. 1995; see review in Webb et al. [2002]). 0.0, 0.0 25.0, 14.0 0.0, 0.0 Initial ENM work examining niche overlap in species 42.4, 97.1 66.9, 88.4 7.6, 75.0 11.8, 50.0 pairs separated by a geographic barrier supported the prediction that niche conservatism characterizes evolutionary diversification (Peterson 2001). However, more RESULTS recent ENM work has suggested that patterns of niche Niche characteristics of members evolution beyond sister taxa can be inconsistent and not of the Anolis sagrei group conserved (Rice et al. 2003). Our results from the The modeled niche of each species served as an environmental-envelope overlap analysis are congruent accurate predictor of the species’ distribution in all cases with these more recent findings. No evidence of (Table 1, Fig. 2). The GARP application provided 10 generalized niche conservatism exists for the A. sagrei ‘‘best’’ models. We used the average accuracy of these group in Cuba. Overall, no relationship was found models for comparisons with WhyWhere model accu- between phylogenetic and niche similarity. This results racy. Prediction accuracy was higher with the Why- because some closely related species have greatly Where algorithm than with GARP for 7 of 11 species divergent niches, whereas some distantly related species (Table 1), thus we used the WhyWhere predictions to are quite similar in their niches. assess individual species pair overlap. A closer look at patterns of niche similarity using the When locality data from one species were compared ENM predictions and species locality data allows a more to the predicted distribution of a sister species using the detailed level of inference. All combinations of phylo- WhyWhere predictions, significant cases of niche con- genetic relatedness and degree of niche overlap are seen servatism are recovered (Table 2). Additionally, several in the data (Table 2). Examples of closely related taxa cases occurred in which distantly related species had with significantly high niche overlap (e.g., A. bremeri–A. niches that are more similar than would be expected by ophiolepis, A. bremeri–A. sagrei, A. mestrei–A. ophiole- chance, whether resulting either from convergence or pis) indicate niche conservatism. Additionally, high stasis (Table 2). There are also cases in which species levels of niche differentiation are seen in close relatives pairs exhibit less overlap than expected by chance; that exhibit significantly little niche overlap (e.g., A. however, whether this limited overlap is due to selection- allogus (east)–A. allogus (west), A. homolechis–A. jubar). driven divergence or random diversification is unclear. In all of these previous scenarios, both members of the sister species pair exhibited the same reciprocal relationship. In addition, in one case, A. quadriocellifer and A. bremeri, the relationship was not reciprocal: although a significant percentage of locality data from A. bremeri does not fall within the A. quadriocellifer prediction, a significant percentage of locality data from A. quadriocellifer does fall with in the A. bremeri prediction. We regard this as indicating that A. quadriocellifer resides in a specialized component of the A. bremeri niche (Fig. 3). The first and second principal components explained 42.4% and 25.7% of the overall variance in the PCA, respectively (Table 3). The Mantel test examination of the relationship between percentage of environmental envelope overlap and phylogenetic similarity indicates FIG. 3. (A) Predicted distribution of A. quadriocellifer with no consistent pattern in the evolution of the species actual localities (points) of A. bremeri. (B) Predicted distribu- niche among species in the A. sagrei group (r ¼ 0.10, P ¼ tion of A. bremeri with actual localities (points) of A. 0.26) (Fig. 4). Indeed, the most closely related taxa show quadriocellifer. S36 JASON H. KNOUFT ET AL. Ecology Special Issue

TABLE 3. PC1 and PC2 loadings from principal-components and Cunningham 2000, Webster and Purvis 2002), we analysis of environmental variables for species in the Anolis have avoided this approach by focusing only on the sagrei group. niches of extant species in the context of their PC1 PC2 phylogenetic similarity. The trade-off of this ‘‘non- Environmental variable loadings loadings directional’’ (sensu Harvey and Pagel 1991) approach, Elevation 0.738 0.061 however, is that we are less able to make statements Mean annual temperature 0.921 0.145 about the direction in which evolution has occurred. In Mean diurnal temperature 0.006 0.731 particular, we have a number of cases in which nonsister Isothermality 0.023 0.061 Temperature seasonality 0.099 0.562 taxa, and indeed sometimes taxa that are only distantly Maximum temperature, warmest month 0.867 0.148 related, have significantly similar niches. Two processes Minimum temperature, coldest month 0.801 0.537 could account for such cases. On the one hand, two Temperature annual range 0.020 0.864 Mean temperature, wettest quarter 0.803 0.337 species might have retained the ancestral niche through Mean temperature, driest quarter 0.837 0.427 the course of time; in other words, their conserved niche Mean temperature, warmest quarter 0.891 0.038 similarity would be an example of evolutionary stasis. Mean temperature, coldest quarter 0.892 0.330 Annual precipitation 0.794 0.027 Alternatively, the two species might have independently Precipitation, wettest month 0.709 0.045 derived the same niche through convergent evolution. Precipitation, driest month 0.501 0.718 Without inferring ancestral niches, these two possibil- Precipitation seasonality 0.301 0.835 Precipitation wettest quarter 0.684 0.216 ities are difficult to distinguish. Precipitation, driest quarter 0.565 0.705 One other interesting situation occurred between the Precipitation, warmest quarter 0.520 0.644 sister taxa A. quadriocellifer and A. bremeri, in which a Precipitation, coldest quarter 0.461 0.796 significant proportion of A. quadriocellifer localities fall within the A. bremeri prediction, whereas the reciprocal pattern is not exhibited. We interpret this to indicate that Many cases of low niche overlap among distantly related the A. quadriocellifer niche is more specialized than, and taxa are also apparent (e.g., A. allogus (east)–A. bremeri, nested within, the A. bremeri niche. Again, however, the A. mestrei–A. rubribarbus). In these cases, examination direction in which this evolutionary change occurred, of species’ niches in a phylogenetic context makes whether from generalist to specialist or vice versa, is evolutionary interpretation, either conservatism or difficult to discern. Nevertheless, the sister taxon to the A. divergence, obvious. By contrast, the evolutionary quadriocellifer–A. bremeri species pair (Fig. 1) is A. sagrei, explanation for similar niches among distantly related a species that has a broad niche similar to that of A. taxa is not so clear-cut: long-term conservatism or bremeri (Table 2). Consequently, the parsimonious con- convergence are both possibilities. clusion is that a broader niche is ancestral and the nar- Although the natural history of many members of the rower niche of A. quadriocellifer is derived in this species. A. sagrei group is poorly studied, there appear to be at Despite methodological differences, our results are least four axes along which the members of this group broadly congruent with previous studies on dendrobatid partition habitat: heliothermy (sun vs. shade-loving), frogs (Graham et al. 2004) and Aphelocoma jays (Rice et forest type (xeric vs. mesic), habitat openness (woodland vs. open habitat), and substrate type (trunks vs. rocks) (Ruibal 1961, Ruibal and Williams 1961, Schwartz and Henderson 1991, Rodrı´guez-Schettino 1999, Losos et al. 2003). Only one of these axes (i.e., forest type) involves a geographic scale of niche partitioning that is appropriate for the methods discussed here. A previous study (Glor 2004) suggested that divergence along this axis, and with respect to substrate type, has occurred repeatedly, perhaps accounting for the observed lack of conservatism. The two other axes (heliothermy and habitat openness) meanwhile, appear more conserved in the sense that they characterize large deeply divergent clades (Glor 2004). Consequently, further study may reveal a greater degree of niche conservatism, particularly along these axes, than we discuss. Our approach differs from previous niche modeling exercises (Rice et al. 2003, Graham et al. 2004) in which phylogenetic methods have been used to infer the niche of hypothetical ancestral taxa. Because in many cases FIG. 4. Relationship between patristic distance and eco- ancestral reconstructions probably have low accuracy logical-envelope overlap in species pairs of the Anolis sagrei (Schluter et al. 1997, Losos 1999, Martins 1999, Oakley group. July 2006 NICHE EVOLUTION IN ANOLIS LIZARDS S37

PLATE 1. Example of a Cuban trunk-ground anole lizard species (Anolis rubribarbus) from Guantanamo Province at Sendero Natural et Recreo de Nibujon, Cuba. Photo credit: R. E. Glor. al. 2003): in all three cases, many instances of closely allopatric speciation and not a consequence of species related taxa that diverge greatly in niche have been distributions actually tracking climatic conditions. This discovered. Thus, the balance of evidence to date can present an interpretive dilemma for sister species provides little consistent evidence that environmental that appear to diverge in niche characteristics. This niches are phylogenetically conservative. The main scenario is displayed by only one species pair in our data counterexample to date is a study of Mexican bird set (A. jubar–A. homolechis). Finally, an important species that showed that allopatric populations on either component of anole community ecology and evolution side of the Isthmus of Tehuantepec tend to exhibit niche is partitioning of habitats (e.g., cool/hot; high/low) conservatism (Peterson et al. 1999, Peterson and Holt within a site (Schoener 1968, Williams 1983, Losos et al. 2003). Although it is tempting to suggest that the focus 2003), a scale of habitat far too small to be detected by on allopatric populations explains these discrepant these sorts of data. Clearly, a next step in niche modeling findings, this conclusion is unwarranted, as closely will be the integration, of broad- and fine-scale niche related allopatric sister taxa show divergence in den- characteristics to elucidate the determinants of local and drobatid frogs and anoles. regional distributions and habitat use. The broad-scale environmental data used in ENM algorithms successfully predict anole occurrence on a ACKNOWLEDGMENTS regional scale. Investigating the role that adaptation to We greatly appreciate the time and efforts of the Curators different climatic niches has played in anole evolution and Collection Managers at the American Museum of Natural History, the California Academy of Sciences, Field Museum, will contribute importantly to understanding the genesis the Museum of Comparative Zoology, Harvard University, the of the incredible diversity of this species-rich clade. Museum of Vertebrate Zoology, University of California at Nonetheless, this approach has limitations. First, the Berkeley, the Smithsonian National Museum of Natural resolution of the GIS data limits analysis to regions, History, and the University of Kansas Natural History rather than specific localities. Thus, this approach can Museum who assisted in providing locality data for species in this study. We also greatly appreciate the efforts of D. Ullman only investigate environmental determinants of regional who georeferenced a large percentage of these data. J. H. co-occurrence, rather than true sympatry. Second, Knouft was supported by a grant from the National Science current species distributions may be a result of recent Foundation (DBI-204144). S38 JASON H. KNOUFT ET AL. Ecology Special Issue

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PHYLOGENY AND THE HIERARCHICAL ORGANIZATION OF PLANT DIVERSITY

1,4 1 1 2 3 JONATHAN SILVERTOWN, MIKE DODD, DAVID GOWING, CLARE LAWSON, AND KEVIN MCCONWAY 1Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes, MK7 6AA UK 2Centre for Agri-Environmental Research, School of Agriculture, Policy and Development, The University of Reading, Earley Gate, P.O. Box 237, Reading, RG6 6AR UK 3Department of Statistics, The Open University, Walton Hall, Milton Keynes, MK7 6AA UK

Abstract. R. H. Whittaker’s idea that plant diversity can be divided into a hierarchy of spatial components from a at the within-habitat scale through b for the turnover of species between habitats to c along regional gradients implies the underlying existence of a, b, and c niches. We explore the hypothesis that the evolution of a, b, and c niches is also hierarchical, with traits that define the a niche being labile, while those defining b and c niches are conservative. At the a level we find support for the hypothesis in the lack of close significant phylogenetic relationship between meadow species that have similar a niches. In a second test, a niche overlap based on a variety of traits is compared between congeners and noncongeners in several communities; here, too, there is no evidence of a correlation between a niche and phylogeny. To test whether b and c niches evolve conservatively, we reconstructed the evolution of relevant traits on evolutionary trees for 14 different clades. Tests against null models revealed a number of instances, including some in island radiations, in which habitat (b niche) and elevational maximum (an aspect of the c niche) showed evolutionary conservatism. Key words: coexistence; community assembly; diversity; evolutionary lability; geographical range; habitat; hydrology; niche overlap; plant community; plant phylogeny.

INTRODUCTION evolutionarily labile. If these traits determine a species’ R. H. Whittaker (1975) proposed that diversity should niche, community structure will appear free of phyloge- be analyzed at a hierarchy of spatial scales. At the local netic conservatism. In contrast, traits that vary little scale, a diversity represents the number of species found among terminals on the same tree indicate that their within a habitat. These species occur in sufficient evolution is likely to be more conservative. Niche-related proximity to interact with one another. At intermediate traits of this kind can potentially produce a phylogenetic scales, b diversity quantifies the turnover in species that signal in the structure of ecological communities. Whether takes place between habitats or along environmental conservatively evolving niche traits actually do produce gradients. At a still wider scale, c diversity is the species this signal depends upon the ecological processes of diversity of a region. Of a and b diversity, Whittaker community assembly that determine how many representatives of a conservatively evolving clade are present. (1975:119) wrote that they ‘‘will be recognized as In areas of high endemism such the Cape Floristic Region consequences of niche differentiation and habitat diver- of South Africa or oceanic archipelagos such as Hawaii, sification of species, respectively.’’ Not long afterwards, some communities might have been assembled, at least in Pickett and Bazzaz (1978) explicitly referred to these part, by adaptive radiation in situ. This is where we might concepts as the a niche and the b niche. (A glossary of expect to find recent evolutionary events influencing terms is given in Table 1.) Although these terms were not community structure most strongly. However, this form widely adopted when they were first introduced, recent of community assembly is a rare event, and most plant research on the phylogenetic structure of ecological communities, including some on islands such as those in communities suggests that the distinction between a and Macaronesia (Santos 2001), have been assembled from b niches should receive greater attention, because the plants with quite disparate phylogenetic histories (Pen- hierarchical relationship between them might reflect the nington et al. 2004, Pennington and Dick 2004). hierarchical structure of evolutionary trees (Fig.1). We ask two central questions. First, do ecological For a given phylogeny, heritable traits that vary freely traits evolve in a conservative manner? Second, is there a among the terminals (tips) of the tree are likely to be difference in evolutionary lability between the traits that underlie a and b niches? Recent studies of the Manuscript received 18 January 2005; revised 9 September phylogenetic distribution of ecological traits have tended 2005; accepted 13 September 2005. Corresponding Editor (ad to emphasize the conservative nature of plant trait hoc): C. O. Webb. For reprints of this Special Issue, see footnote 1, p. S1. evolution and suggested that this influences community 4 E-mail: [email protected] assembly (Tofts and Silvertown 2000, Webb 2000, S39 S40 JONATHAN SILVERTOWN ET AL. Ecology Special Issue

TABLE 1. Deﬁnitions of terms used in the text.

Term Definition Source a niche The region of a species’ realized niche corresponding to species diversity at the local (a) scale 2, 5 where interactions among species occur b niche The region of a species’ niche that corresponds to the habitat(s) where it is found; equivalent to 2, 3 the ‘‘habitat niche’’ Grubb (1977) c niche The geographical range of a species 6 Community The collection of species that predictably co-occurs within a particular type of habitat Habitat The kind of environment where a species occurs, defined largely by physical conditions; note 2 that conditions will usually be influenced by organisms as well as physical factors, but direct interactions among organisms are not used to define habitats Lability The property of evolutionary changeability in a trait Niche An n-dimensional hypervolume defined by axes of resource use and/or environmental conditions 1, 4 and within which populations of a species are able to maintain a long-term average net reproductive rate 1 Niche trait A measurable property of a species, by which its niche (a, b,orc ) can be defined Realized niche The region of its niche that a species is able to occupy in the presence of interspecific 1 competition and natural enemies Sources: 1, Hutchinson (1957); 2, Whittaker (1975); 3, Pickett and Bazzaz (1978); 4, Chase and Leibold (2003); 5, Silvertown (2004); 6, Silvertown et al. (2006).

Prinzing et al. 2001, Webb et al. 2002, Ackerly 2003, corollary of this is that a niches and coexistence will 2004, Chazdon et al. 2003). One example of this pattern necessarily be determined by labile traits. In short, is the long-standing observation that, in many commun- Silvertown et al. (2006) proposed that competing species ities, there is a higher ratio of species per genus than must share b niches in order to occur in the same habitat, would be expected if communities were assembled by but they must have different a niches in order to coexist. random draws from the species pool (e.g., Williams Silvertown et al. (2006) proposed that, by extension of 1964). If congeneric species are overrepresented in the relationship between a and b niches and Whittaker’s communities, then it follows that they must share (1975) a and b diversity, the geographical range of a ecological traits that influence community assembly species can be regarded as its c niche. Thus, there is a and that these traits evolve more slowly than the rate hierarchy of three niche levels with c at the top (Fig. 1). of appearance of new species. The little evidence that is so far available suggests that b Other studies, however, suggest that some traits that niche traits are evolutionarily conservative; data pertain- influence community structure do not evolve conserva- ing to the evolution of the c niche are even more sparse. tively. Cavender-Bares et al. (2004) detected labile evolu- Prinzing et al. (2001) analyzed the niches of European tion in the soil moisture tolerances of North American plant species using Ellenberg indicator values (Ellenberg oak species and found that these species segregated along 1979, Ellenberg et al. 1991) and found strong evidence of soil moisture gradients. Silvertown et al. (1999) found evolutionary conservatism. These values were devised to that plant species in English meadow grasslands also quantify on an ordinal scale where different plant species segregated on hydrological gradients and later reported are found in central Europe along major environmental that there is no correlation between the ecological dis- axes, such as soil moisture, pH, light, and soil fertility. tance between species in hydrological niche space and Several studies have found that Ellenberg values are their phylogenetic distance as measured by the evolution stable traits that consistently predict the b niche of of the rbcL gene (Silvertown et al. 2006). How can these data be reconciled with the many other examples of the species across Europe more generally (Thompson et al. conservative evolution of ecological traits? 1993, Hill et al. 2000, Schaffers and Sykora 2000, Silvertown et al. (2006) suggested that the apparent Prinzing et. al. 2002). Ellenberg values can be regarded contradiction between the lack of phylogenetic signal in as b niche traits, because they refer to large-scale their data, which implies evolutionary lability in hydro- environmental gradients. However, since a niches are logical niches, and contrary findings by other authors nested within b niches, some correlation between traits implying conservative evolution in some traits could be like soil moisture tolerance is to be expected. explained if the traits have different evolutionary lability. Ackerly (2004) examined phylogenetic conservatism They proposed that habitat-determining traits that in the evolution of leaf traits that are associated with influence b diversity, and which may be said to define adaptation to Mediterranean climates in California the b niche (Pickett and Bazzaz 1978), evolve conserva- chaparral habitat. These are a good example of traits tively. By contrast, traits involved in coexistence and that associated with the b niche. Specific leaf area was influence a diversity, defining the a niche, are evolutio- significantly conserved in all four families analyzed, and narily labile. Such a pattern could arise if, as most leaf size in three. These results suggest that sclerophylly theories of coexistence demand (Chesson 2000), species and other leaf traits associated with Mediterranean must differ from each other in order to coexist. The habitats evolved before California chaparral was colo- July 2006 HIERARCHY OF PLANT DIVERSITY S41 nized, supporting the view that the b niche traits evolve in a conservative manner. Very little evidence is available concerning the evolution of c niches. Qian and Ricklefs (2004) found that the latitudinal ranges, and hence c niches, of 57 plant genera with disjunct distributions in North America and Asia were correlated between continents, suggesting that the genera had highly conserved c niches that dated to before the origin of the disjunctions, perhaps 18 million years ago in the case of woody species. How typical this result will prove to be of c niches in general is not clear at present. Other studies have examined the degree of range overlap between members of the same clade (Barra- clough and Vogler 2000, Graham et al. 2004). Barra- clough and Vogler (2000) found that in a range of vertebrate and insect phylogenies range overlap was low between recently diverged taxa, but increased with time since divergence. This indicates that speciation occurs more often in allopatry (no range overlap) than sympatry, but does not directly address the issue of c niche evolution, since ranges can be split (i.e., become allopatric) by the appearance of environmental barriers, without any need for evolutionary change in the c niche. FIG. 1. A Venn diagram showing the nested hierarchy of a, b, and c niches superimposed upon a hypothetical phylogenetic In this paper we present four new lines of evidence tree. Note that the rectangles representing each kind of niche that have a bearing on the evolutionary conservatism of intersect the phylogenetic tree at progressively deeper levels a, b, and c niche traits. Each piece of evidence applies a from a to b to c niches, indicating earlier origin and greater different kind of test, as appropriate to the data conservatism. available. First, we take a closer look at the English wet meadow evolution is conservative. As a subsidiary hypothesis, the communities in which Silvertown et al. (2006) found a expected patterns of conservatism should be stronger in niche traits to be evolutionarily labile. We perform a clades that have evolved in continental areas, where new analysis of the data in which we ask whether groups available habitats are likely to have been already oc- of specialists that are confined to particular subcom- cupied by competitors, than in clades that have radiated munity types are more closely related to one another on islands where most habitats were unoccupied. than would be expected for randomly drawn samples Finally, we apply the same kind of test to c niche from the same community. A smaller phylogenetic evolution by optimizing maximum elevation (reflecting distance between specialists than between randomly the c niche) onto phylogenies for two clades. drawn nonspecialists would imply evolutionary conservatism in the specialist group. METHODS Second, we reanalyze published data on various Are habitat specialists phylogenetically clustered? ecological traits in a number of plant communities to determine whether a niche overlap between congeners is Silvertown et al. (1999, 2001) previously analyzed the greater or less than between noncongeners in the same niche relationships of species in two mesotrophic grass- community. Although we recognize that there is no land (meadow) plant communities classified as MG5 and consistent phylogenetic definition of a genus, and that MG8 by the British National Vegetation Classification some may be very old, congeners can usually be expected (Rodwell 1992). Silvertown et al. (2001) suggested that to be more closely related than species from other genera some of the niche separation observed in these drawn from the same community. Trait variation ought communities arose as deep as the split between mono- therefore to be smaller between congeners than non- cots and eudicots, indicating that niche specialization congeners for conservatively evolving traits that predate occurs within particular clades. For the present study, the origin of the genus, but similar for labile traits. we identified specialists from within each of the two Third, we conduct a test of a prediction derived from community types using data from an extensive survey the hypothesis that b niche evolution is conservative by made by Gowing et al. (2002). This survey recorded an examining the number of inferred transitions in habitat estimate of percent cover of all species present in 3904 1 (reflecting the b niche) within plant phylogenies for a 3 1 m quadrats across 18 sites representative of MG5, sample of 12 independent clades. Changes of habitat MG8, and other floodplain hay meadow types in should be fewer than expected by chance if b niche England. Quadrats were classified into 12 communities S42 JONATHAN SILVERTOWN ET AL. Ecology Special Issue

TABLE 2. Comparison of a niche overlaps between congeneric pairs of herb species in nine genera and mean overlaps between the congeners and species in other genera.

Total no. Overlap Overlap overlapping with with Data Genus species congeners Comparison noncongeners a niche axes source Drosera 9 0.00 , 0.60 water table gradient in a bog community 5 Prosopis 16 0.15 , 0.38 soil moisture and nutrients in a desert community 4 Dentaria 17 0.42 . 0.37 forest understory microtopography and light 1 Galium 17 0.45 ¼ 0.45 forest understory microtopography and light 1 Senna 16 0.48 . 0.31 soil moisture and nutrients in a desert community 4 Trillium 17 0.51 , 0.52 forest understory microtopography and light 1 Aster 10 0.76 . 0.44 phenology and microtopography in forest understory 2 Helictotrichon 10 0.87 . 0.83 shoot phenology in grassland 3 Festuca 10 0.88 . 0.83 shoot phenology in grassland 3 Notes: There is no significant difference overall between the degree of overlap found between congeners and the overlap between noncongeners (Wilcoxon matched-pairs test, Z ¼ 0.42, P ¼ 0.67). Sources: 1, Mann and Shugart (1983); 2, Beatty (1984); 3, Sydes (1984); 4, Shaukat (1994); 5, Nordbakken (1996). and subcommunities using the program TWINSPAN run for each null model. If specialists are significantly (Hill 1979). clustered phylogenetically, then the mean and variance of A group of seven species characteristic of the MG5a pairwise rbcL distances should fall in the lower 5% of subtype of the MG5 community consisted of Trifolium values in the null distribution of each statistic. pratense (Fabaceae), Rhinanthus minor (Scrophularia- a niche overlap among congeners vs. other species ceae), Dactylis glomerata (Poaceae), Prunella vulgaris (Lamiaceae), Heracleum sphodylium (Apiaceae), and Through an extensive review of the literature on plant Leucanthemum vulgare and Leontodon saxatilis (both niches, we identified five studies of plant communities Asteraceae). Within the MG8 community type, 10 from which it was possible to compute a niche overlaps species were identified as specialists associated with a within and between genera. There were nine sets of Carex disticha subcommunity. These were Carex dis- congeners in total. The validity of generic names was ticha, C. distans, and Eleocharis uniglumis (Cyperaceae); checked against the online versions of Clayton and Senecio aquaticus and Bellis perennis (Asteraceae); Williamson (2003) for grasses and Brummitt (1992) for Juncus inflexus, J. articulatus,andJ. subnodulosus other species. A name change affected one genus (Juncaceae); Festuca arundinaceae (Poaceae), and Trifo- (Dentaria to Cardamine), but did not alter the implied lium fragiferum (Fabaceae). Specialists occurred more evolutionary relationships between this genus and the frequently in the designated subcommunity types rest of the community with which it was compared. (MG5a, MG8 C. disticha) than in any other of the 12 Niche axes varied between studies (Table 2), but overlap communities identified in the TWINSPAN analysis. was measured using Pianka’s index in all cases (Pianka Phylogenetic distances between all pairwise combina- 1973). The pairwise overlap, Ojk, between the niche of tions of 52 species belonging to MG5 and MG8 species j and the niche of species k is communities were calculated by Silvertown et al. P pijpik (2006). Distances were calculated as the sum of branch Ojk ¼ qPffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð1Þ 2 2 lengths connecting species in a tree fitted to rbcL pijpik sequences using maximum likelihood in PAUP* (Swof- ford 1996). For each of the two specialist groups, we for all resource states, i. In Eq. 1, pij is the proportion of computed the mean and variance of pairwise phyloge- total resources used by j that consist of resource state i, netic distances among members of the group and and pik is the proportion of total resources used by k that compared these with expected (null) distributions pro- consist of resource state i. Values of Ojk range from 0 to duced by randomization. Null distributions were derived 1. The difference in mean overlap between congeners, by sampling groups of n species at random from the 52 and between congeners and the rest of the community, species in the meadow species pool for which rbcL was tested by a Wilcoxon matched-pairs test (Sokal and sequences are known, where n was the number of species Rohlf 1995). in the specialist group. To avoid bias in the species pool b and c niche transitions caused by underrepresentation of sequences for Carex and Juncus, we added extra copies of rbcL sequences for We conducted a search of articles and citations in species in these genera when conducting the test on the American Journal of Botany, Systematic Botany, and TreeBASE (University of Buffalo, New York, USA; Carex disticha subcommunity type. Using substitutes in available online)5 to identify molecular phylogenetic this way does not introduce bias, because rbcL sequence differences among species of Carex and among Juncus species are very small. A total of 104 randomizations were 5 hwww.treebase.orgi July 2006 HIERARCHY OF PLANT DIVERSITY S43 studies of plants in which 50% of the extant species in a a niche overlap among congeners vs. other species clade had been sampled (Tables 3 and 4). Phylogenies Table 2 compares a niche overlaps between congene- with ,20 species were excluded because randomization ric pairs of species in nine genera with mean overlaps tests of the kind we used to detect phylogenetic between the congeners and species in other genera. conservatism have low statistical power with sample There is no significant difference overall between the sizes below this limit (Blomberg et al. 2003). Habitat and degree of overlap found between congeners and the elevational data were obtained from the same source as overlap between noncongeners (Wilcoxon matched- the phylogeny wherever they were given, or from pairs test, Z ¼ 0.42, P ¼ 0.67). standard floras where they were not (Tables 3 and 4). Habitat is by definition a b niche trait. We treated b and c niche transitions elevational maximum as a c niche trait, because it Table 3 presents b niche transitions in seven island and delimits the vertical dimension of a species’ range and is five continental clades. There was significant conserva- clearly related to climate. tism in the evolution of the b niche in five of the seven Evolution of habitat and elevational maximum (EM) island clades and in three of the six continental cases. were optimized onto trees using MacClade 3.06 (Mad- Habitat factors associated with conservatism included dison and Maddison 1992). Habitat was treated as a the six major altitudinal zones in the Canary Islands (the polymorphic character for species that were present in principal archipelago of Macaronesia) in the case of the more than one habitat type. Elevational maximum was Aeonium clade, but not in the Echium or Argyranthemum scored as a categorical variable with four classes: 0, EM clades. When a binary coding of b niche into dry vs. 1000 m; 1, 1000 m , EM 2000 m; 2, 2000 m , EM mesic was used, Argyranthemum and Sonchus did show 3000 m; 3, EM . 3000 m. Tests for phylogenetic conservatism, but Aeonium and Sideritis did not (Table conservatism were performed by comparing the number 3). Similar patterns were found in Hawaii, with species in of transitions (steps) between habitat or EM states the Schiedea clade showing conservative evolution in required to account for the observed distribution of respect of eight habitat types; this clade and the habitats among terminal taxa with a null distribution. silversword alliance showed fewer transitions than We obtained a null distribution for the number of expected between wet and dry environments (Table 3). habitat or EM transitions to be expected in any given In continental clades, conservatism occurred in both tree by randomly shuffling the observed states among habitat variables (serpentine soils and forest vs. open its terminals (Maddison and Slatkin 1991). Using habitats) analyzed in Calochortus, in one of three MacClade 3.06, we performed 103 randomizations for variables (occurrence in vernal pools) in Mimulus, and each tree. The probability that an observed number of in preference among four habitat types in Narcissus steps occurred by chance was the frequency of (Table 3). Neither Linanthus nor Primula clades showed transitions of the same or smaller value found in the evidence of b niche conservatism. null distribution. Frequencies ,0.05 were treated as Of the two clades analyzed for c niche conservatism, evidence of significant conservatism in the evolution of EM evolved conservatively in Pinus, but not in Mimulus habitat preference or EM. The randomization test we (Table 4). Whether the data were coded as four EM used is normally employed on binary characters, but classes or two did not affect either outcome. some of our tests involved more than two niche DISCUSSION categories (e.g., four EM classes of the c niche). In order to test the robustness of our results against the Collectively, the analyses performed here demonstrate unconventional use of multistate characters, where a lack of phylogenetic signal in the ecological structure variables could be combined on the basis of some of communities, but, in contrast, indicate its presence in ecological variable (e.g., dry vs. mesic), we ran tests on at least some instances of how speciation populates data recoded as a single binary character. different habitats and how elevational range evolves. The results support the suggestion that a niche traits are RESULTS evolutionarily labile, while b and c niche traits might evolve in a more conservative manner. However, there Are habitat specialists phylogenetically clustered? are caveats. The mean and variance of pairwise rbcL distances The species in the samples used to examine ecological among the seven specialists in the MG5a community structure in communities on the one hand and adaptive were 0.112 (P ¼ 0.144) and 0.0017 (P ¼ 0.226), radiation among habitats and elevations on the other respectively. For the 10 specialists in the Carex disticha were differently constituted. In the first instance, we community, the mean and variance were respectively measured phylogenetic distances between a collection of 0.125 (P ¼ 0.428) and 0.0023 (P ¼ 0.362). In neither species that had passed through the various ecological community was the mean or the variance significantly filters involved in community assembly. This resulted in lower than expected by chance; thus the null hypothesis an extremely rarefied sampling of disparate branches of of no phylogenetic clustering among specialists cannot the angiosperm phylogeny, including monocot and be rejected. eudicot clades. It would be necessary to analyze a less S44 JONATHAN SILVERTOWN ET AL. Ecology Special Issue

TABLE 3. Characteristics and numbers of observed b niche (habitat) transitions in island and continental clades estimated by phylogenetic optimization, along with the number expected from a null model.

Clade Region Sample/Clade size Types of b niche (no. habitats) Island clades Aeonium, Greenovia, and Macaronesia 51/63 cliffs and rocks, xerophytic scrub, Monanthes thermophile woodland, laurel forest, pine forest, subalpine (6) dry, mesic (2) Argyranthemum Macaronesia 51 pops/23 spp. cliffs and rocks, xerophytic scrub, thermophile woodland, laurel forest, pine forest, subalpine (6) dry, mesic (2) Echium Macaronesia 21/27 cliffs and rocks, xerophytic scrub, thermophile woodland, laurel forest, pine forest, subalpine (6) dry, mesic (2) Sideritis Macaronesia 32/32 dry, mesic (2) Sonchus Macaronesia 31/34 dry, coastal, mesic (3) dry and coastal, mesic (2) Schiedea and Hawaii 30/28 dry forest, dry shrubland, dry cliffs, Alsinidendron dry subalpine, shrubland, diverse mesic forest, wesic forest, wet forest (8) dry, mesic (2) Silversword alliance Hawaii 36/36 dry, mesic (2) Continental clades Calochortus Western North America 67/67 serpentine, other habitats (2) forest, open habitats (2) Narcissus Europe 23/27 dry rocky open Mediterranean hillsides, montane wet meadows, oak woodland, lowland mesic Mediterranean (4) Linanthus, Leptosiphon clade California 28/28 woodland and chaparral, grassy areas, serpentine, desert, drying areas in conifer forest (5) dry, other habitats (2) serpentine, other habitats (2) Mimulus Northwestern North America 88/;114 vernal pool, other habitats (2) serpentine, other habitats (2) Primula sect. Auricula Alps 25/25 limestone, acid substratum (2) cliffs and rocks, turf, woodland, alpine/subalpine/tundra (4) *P 0.05, **P 0.01, ***P 0.001. Sample/Clade size is the ratio of the no. species (or, in the case of Argyranthemum, no. populations) in the phylogenetic analysis to the estimated no. extant species that belong to the clade. à The expected number of transitions shown is the mode of the distribution of 1000 runs of the null model. § P values are for the difference between the number of expected transitions and the number of observed transitions (number observed not exceeding number expected). Sources: 1, Hickman (1993); 2, Baldwin and Robichaux (1995); 3, Wagner et al. (1995); 4, Weller et al. (1995); 5, Bohle et al. (1996); 6, Francisco-Ortega et al. (1996); 7, Kim et al. (1996); 8, Barber et al. (2000); 9, Bell and Patterson (2000); 10, Bramwell and Bramwell (2001); 11, Mort et al. (2002); 12, Beardsley et al. (2004); 13, Graham and Barrett (2004); 14, Patterson and Givnish (2004); 15, Zhang et al. (2004); 16, S. C. H. Barrett, personal communication. rarefied sample if we were asking a solely evolutionary cannot be ruled out as a source of the opposing results question, but the following question is specific and (Gotelli and Graves 1996). It may also be that the much ecological: ‘‘Are specialist members of MG5a commun- larger species pool for tropical forest (n ¼ 312) than for ities phylogenetically clustered?’’ In this case, the English meadows (n ¼ 52) makes phylogenetic structure method we have used is appropriate and it gives the more likely to occur or easier to detect among specialists. unequivocal answer, ‘‘no.’’ The approach used to compare niche overlap among It is interesting that Kembel and Hubbell (2006) found congeners with that among other species does not raise an absence of phylogenetic structure in the overall phylogenetic sampling issues, but it does assume that a tropical forest community in the 50-ha plot at Barro niche dimensions relevant to coexistence have been Colorado Island, but that phylogenetic structure did correctly identified. In each case the dimensions occur within specific habitats. Our null model and those measured do seem likely to fulfil this assumption (Table of Kembel and Hubbell (2006) were different, and this 2), but as yet very few field studies of putative plant July 2006 HIERARCHY OF PLANT DIVERSITY S45

TABLE 3. Extended. used specific leaf area (SLA) as a proxy measure of the a niche in Ceanothus and found that this diverged earlier than their climatically defined measure of the b niche. No. b niche transitions Data This finding is at odds with our hypothesis that the a Observed Expectedà P§ sources niche is more labile than the b niche (Fig. 1).

20 25 0.003** 10, 11 b niche transitions Oceanic islands and island-like habitats, such as 10 11 0.303 vernal pools and serpentine barrens in California, 21 24 0.071 6 contain multiple radiations that provide replicates for the test of b niche conservatism. There are several 6 10 0.001*** examples in the endemic flora of vernal pools in the 11 12 0.485 5, 10 California Floristic Province (CFP). An extreme case is the monophyletic genus Downingia that contains 13 species (Schultheis 2001), all but one of which occur in 5 6 0.272 13 13 0.681 8 vernal pools (Ayers 1993). In section Navarretia of the 8 11 0.020* 7 genus Navarretia, four vernal pool species form a clade 6 9 0.036* that is sister to a species that is facultatively associated 18 21 0.019* 3, 4 with the same habitat (Spencer and Rieseberg 1998). In the much larger genus Mimulus, there are roughly six vernal pool species, and four of them are concentrated in 6 9 0.020* 5 8 0.011* 2 one small clade, indicating significant conservatism in this genus, as well (Thompson 1993, Beardsley et al. 2004) (Table 3). 10 15 0.011* 14 13 17 0.049* Also in the CFP, significantly conservative evolution 8 12 0.003** 13, 16 of serpentine tolerance is found in the large genus Calochortus, where seven of a total of 18 species occurring on serpentine soils belong to a single clade 17 18 0.284 1, 9 (Patterson and Givnish 2004) (Table 3). Serpentine species in Mimulus show slight, though non-significant 6 8 0.092 phylogenetic association (Table 3). Phylogenetic rela- 3 3 1.000 tionships among Mimulus species are well resolved, but 3 6 0.002** 1, 12 the weak evidence of phylogenetic conservatism might 5 6 0.156 5 7 0.120 15 easily be strengthened by more ecological data. Just 12 12 0.641 three of 28 species in the Leptosiphon clade of the genus Linanthus occur on serpentine, but they represent three independent evolutionary events (Patterson 1993, Bell and Patterson 2000), so there is no evidence of niches have proven their role in coexistence beyond all conservatism in this case. doubt (Silvertown 2004). The result of the congeners Kelch and Baldwin (2003) compared the mean genetic test performed here is consistent with large a niche divergence measured at ITS and ETS rDNA loci among differences that have been found between sympatric terminal taxa in seven clades that have evolved within species in, among others, the following genera: Acer the CFP, in addition to the cases already mentioned. (Sipe and Bazzaz 1994, 1995), Adenostoma (Redtfeldt There was a positive relationship between genetic and Davis 1996), Dryobalanops (Itoh et al. 2003), divergence within a clade and the number of plant Macaranga (Davies 2001), Piper (Fleming 1985), Psy- communities in which its members are found. A clade of chotria (Valladares et al. 2000), Quercus (Cavender- Cirsium species endemic to the CFP was an outlier from Bares et al. 2004), Ranunculus (Harper and Sagar 1953), the relationship as a whole, inhabiting a greater variety Salix (Dawson 1990), and Typha (Grace and Wetzel of plant communities than would be expected for the 1981). It is clear that coexisting congeners are often as degree of genetic divergence among its members. This ecologically different from each other as they are from deviation could result either from an abnormally high unrelated members of the same communities. This rate of evolutionary shifts between habitats in the CFP implies that a niche traits are evolutionarily labile, Cirsium clade, or an abnormally low rate of molecular although proof of this requires evolutionary changes to evolution. High ecological diversity relative to rDNA be analyzed against an explicit, and preferably dated, variation also occurs in the larger North American phylogeny. Cirsium clade of which the CFP endemics form one part Ackerly et al. (2006) tested the order in which a and b (Kelch and Baldwin 2003). This could indicate that the niche traits evolved in the shrub genus Ceanothus. They evolutionary lability of habitat depends on lineage. S46 JONATHAN SILVERTOWN ET AL. Ecology Special Issue

TABLE 4. Characteristics and no. c niches, along with no. b niche transitions, based upon upper elevational maximum in two large clades estimated by phylogenetic optimization

No. b niche transitions Sample/Clade No. Data Clade Region size c niches Observed Expected P sources Mimulus Northwestern North America 88/;114 4 27 27 0.399 2, 3 2 19 19 0.680 Pinus Europe, Asia, North and Central America 101/;120 4 29 37 0.003 1, 4 2 13 19 0.003 Notes: Data were analyzed for c niches coded into four (0–999 m, 1000–1999 m, 2000–3000 m, .3000 m) and two (,2000 m vs. .2001 m) elevational maximum classes (i.e., c niches). Also see Table 3 footnotes for further explanatory details. Sources: (1) Mirov 1967, (2) Hickman 1993, (3) Beardsley et al. 2004, (4) Gernandt et al. 2005.

Radiations on islands also show a mixed picture, two subgenera diverged, which dates it to the deepest although conservatism here is more evident than might node in the phylogeny of Pinus (Gernandt et al. 2005). have been expected given the extreme evolutionary Extant members of the genus comprise a mixture of lability of plant form that is present in Aeonium ancient and quite recently evolved species (Farjon (Jorgensen and Olesen 2001), Sonchus (Kim et al. 1996), so conservatism in their elevational distribution 1996), the silversword alliance (Baldwin and Robichaux cannot simply be attributed to the lack of recent 1995), and other island endemics. The Hawaiian mints speciation. are another endemic group in which considerable morphological variation among species occurs within a Why should a, b, and c niches evolve restricted range of climatic conditions (Lindqvist et al. with different degrees of lability? 2003). It should be recognized that the crude distinction All theories of coexistence based upon nonneutral between wet and dry habitats used for the silverswords processes require that species have different a niches in in Table 3 does not do justice to the enormous range of order to coexist (Chesson 2000). Silvertown et al. (2006) soil moisture conditions present in different habitats in argued that, for this reason, community assembly will Hawaii. The Hawaiian lobeliods are a group that have create structure based upon labile traits. (It will not do radiated across the entire soil moisture gradient so if neutral processes dominate community assembly.) (Givnish et al. 2004). Nonetheless, the unexpected The argument is not that competitive exclusion forces a presence of conservatism of habitat evolution in several niches to evolve in a labile manner, but rather that it island radiations is remarkable. It suggests that speci- prevents any traits that might, for whatever reason, not ation often involves interisland colonization between be evolutionarily labile from facilitating coexistence. similar habitats (Francisco-Ortega et al. 1996) and that Nonlabile traits are prevented from defining the a niche conservative habitat evolution is not confined to by default. Webb et al.’s (2006) study of the effect of continental radiations. interspecific relatedness on seedling mortality implies that apparent competition mediated by disease, as well c niche transitions as direct competition, could cause related species that The distinction between b and c niches is not clear- are insufficiently different to exclude one another at the cut, but neither should we expect it to be. The three local scale. niche types of a, b, and c are segments in Hutchinson’s A filtering process might also operate upon the traits (1957) n-dimensional hypervolume and are bound to that define the b niche, but with opposite effect. overlap along some dimensions. On some dimensions Coexisting species must by definition occupy the same they may be nested, on others they may not. For habitat and must therefore have b niches that overlap. example, since elevation and habitat are closely Thus, the b niche might come to be defined by nonlabile correlated in Macaronesia (Bramwell and Bramwell traits. 2001), conservative evolution of habitat in Aeonium and The filtering processes that could determine the Sonchus (Table 3) also implies conservative evolution of lability of the a niche and the conservatism of the b elevational distribution. We analyzed elevational max- niche do not as easily explain the conservatism of c imum in Mimulus, where its evolution was not niches, such as the latitudinal ranges of woody plants conservative and in Pinus where it was (Table 4). with disjunct distributions (Qian and Ricklefs 2004). For Differences in elevational distribution between the pines c niches, we must invoke either phylogenetic constraint, of different regions of the world were noted by Mirov such as a lack of appropriate genetic variation, or (1967). Grotkopp et al. (2004) found that species in the phylogenetic niche conservatism (PNC) (Harvey and subgenus Pinus occupied significantly lower elevations Pagel 1991). Although the result of stabilizing selection, than those in subgenus Strobus. This implies that it is not clear why PNC should operate with particular elevational distribution has been conserved since the effect on the c niche; we therefore offer a third July 2006 HIERARCHY OF PLANT DIVERSITY S47 explanation. If one thinks of the c niche as being a Barton, N. 2001 Adaptation at the edge of a species’ range. geographical area with climatically defined boundaries, Pages 3–20 in J. Silvertown and J. Antonovics, editors. then the problem of why it evolves conservatively is Integrating ecology and evolution in a spatial context. Blackwell, Oxford, UK. closely allied to another evolutionary question: what Beardsley, P. M., S. E. Schoenig, J. B. Whittall, and R. G. prevents species at range boundaries from evolving the Olmstead. 2004. Patterns of evolution in western North ability to escape beyond those boundaries? Haldane American Mimulus (Phrymaceae). American Journal of (1956) proposed the following answer to this question: Botany 91:474–489. Beatty, S. W. 1984. Influence of microtopography and canopy Adaptation at range boundaries, which is necessary for species on spatial patterns of forest understory plants. spread to be possible, might be genetically constrained Ecology 65:1406–1419. by the swamping effect of gene flow from individuals in Bell, C. D., and R. W. Patterson. 2000. Molecular phylogeny the hinterland that are not adapted to condition at the and biogeography of Linanthus (Polemoniaceae). American Journal of Botany 87:1857–1870. boundary. This process requires that populations at the Blomberg, S. P., T. Garland, and A. R. Ives. 2003. Testing for periphery of a distribution exist as demographic sinks phylogenetic signal in comparative data: Behavioral traits are that require an input of migrants for persistence more labile. Evolution 57:717–745. (Kirkpatrick and Barton 1997, Barton 2001). This is a Bohle, U. R., H. H. Hilger, and W. F. Martin. 1996. Island condition that can be tested. colonization and evolution of the insular woody habit in Echium L (Boraginaceae). Proceedings of the National In this paper we have developed earlier ideas that the Academy of Sciences (USA) 93:11740–11745. hierarchical organization of plant diversity at the a, b, Bramwell, D., and Z. Bramwell. 2001. Wild flowers of the and c scales proposed by Whittaker (1975) corresponds Canary Islands. Rueda, Madrid, Spain. to a hierarchical set of niches. The traits that define the a Brummitt, R. K. 1992. Vascular plant families and genera. Royal Botanic Gardens, Kew, Surrey, UK. hhttp://www. niche appear to be evolutionarily labile, whereas the rbgkew.org.uk/data/vascplnt.htmli phylogenetic evidence suggests that the b niche evolves Cavender-Bares, J., D. D. Ackerly, D. A. Baum, and F. A. in a conservative manner. Perhaps most conservative of Bazzaz. 2004. 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NICHE EVOLUTION AND ADAPTIVE RADIATION: TESTING THE ORDER OF TRAIT DIVERGENCE

1,4 2 3,5 D. D. ACKERLY, D. W. SCHWILK, AND C. O. WEBB 1Department of Integrative Biology, University of California, Berkeley, California 94720 USA 2U.S. Geological Survey/WERC, Sequoia and Kings Canyon Field Station, Three Rivers, California 93271 USA 3Department of Ecology and Evolution, Yale University, New Haven, Connecticut 06511 USA

Abstract. In the course of an adaptive radiation, the evolution of niche parameters is of particular interest for understanding modes of speciation and the consequences for coexistence of related species within communities. We pose a general question: In the course of an evolutionary radiation, do traits related to within-community niche differences (a niche) evolve before or after differentiation of macrohabitat affinity or climatic tolerances (b niche)? Here we introduce a new test to address this question, based on a modification of the method of independent contrasts. The divergence order test (DOT) is based on the average age of the nodes on a tree, weighted by the absolute magnitude of the contrast at each node for a particular trait. The comparison of these weighted averages reveals whether large divergences for one trait have occurred earlier or later in the course of diversification, relative to a second trait; significance is determined by bootstrapping from maximum-likelihood ancestral state reconstructions. The method is applied to the evolution of Ceanothus, a woody plant group in California, in which co-occurring species exhibit significant differences in a key leaf trait (specific leaf area) associated with contrasting physiological and life history strategies. Co- occurring species differ more for this trait than expected under a null model of community assembly. This a niche difference evolved early in the divergence of two major subclades within Ceanothus, whereas climatic distributions (b niche traits) diversified later within each of the subclades. However, rapid evolution of climate parameters makes inferences of early divergence events highly uncertain, and differentiation of the b niche might have taken place throughout the evolution of the group, without leaving a clear phylogenetic signal. Similar patterns observed in several plant and animal groups suggest that early divergence of a niche traits might be a common feature of niche evolution in many adaptive radiations. Key words: adaptive radiation; Ceanothus; Cerastes; Coast Range; community assembly; Euceanothus; habitat, niche conservatism; phylogenetic comparative methods; specific leaf area; Sierra Nevada; trait divergence; Transverse Range.

INTRODUCTION of species distributions. At large spatial scales, species can occupy different macrohabitats or climatic enve- Ecologists have long considered niche differences lopes; the resulting distributions will be largely allo- among species to be essential for species coexistence patric, or, if they do overlap geographically, individuals (Chesson 2000, Chase and Leibold 2003; but see of the two species would rarely encounter one another Hubbell [2001]). The evolution of niche differences due to habitat differentiation. At smaller scales, related among closely related species has received particular species that co-occur in local communities usually attention. Because close relatives tend to be ecologically exhibit spatial or temporal differentiation in micro- similar in many respects (Darwin 1859, Felsenstein habitat, resource use, diet, or other factors. It is at this 1985, Harvey and Pagel 1991, Webb et al. 2002), those local scale, where the balance of intra- and interspecific features that do diverge during speciation will provide interactions influences coexistence and community important insights into ecological differentiation and structure, that the niche concept has played the most consequences for coexistence of closely related species. important role. Following Pickett and Bazzaz (1978) It is useful in this context to distinguish two scales of and Silvertown et al. (2006), we employ the term a niche niche differentiation, corresponding to different scales to describe these small-scale components of the niche that differ among co-occurring species, corresponding Manuscript received 21 January 2005; revised 9 August 2005; to Whittaker’s (1975) use of a diversity for diversity of accepted 11 August 2005. Corresponding Editor: A. A. local communities. In contrast, the b niche is defined as Agrawal. For reprints of this Special Issue, see footnote 1, p. macrohabitat and climate factors related to larger scale S1. distributions, corresponding to the b component of 4 E-mail: [email protected] 5 Present affiliation: Arnold Arboretum of Harvard Uni- diversity among habitats in a landscape. In this paper, versity. we do not distinguish the proposed b and c niche, which S50 July 2006 NICHE EVOLUTION AND ADAPTIVE RADIATION S51 refers to distributions at the scale of habitat vs. Recent developments of phylogenetic methodology geographic range (Silvertown et al. 2006), as these have offer outstanding opportunities to reevaluate these equivalent implications in terms of species interactions classic questions. Here, we present a comparative at the community level. approach to the problem of niche evolution by It has been argued that species within local commun- introducing a new comparative method designed to test ities tend to be phylogenetically overdispersed; i.e., the relative timing of divergence in two ecological traits closely related species co-occur less than expected, (e.g., a vs. b niche axes); we then use the test to evaluate relative to an appropriate null model (Elton 1946, the sequence of trait divergence in the radiation of the Williams 1964, Gotelli and Graves 1996, Cavender- woody plant group Ceanothus in California. Our results Bares et al. 2004). This pattern would suggest that a indicate that a niche traits related to local scale niche parameters are evolutionarily conserved and/or b coexistence diverge first in the radiation of this group, niche parameters are highly divergent, such that close a pattern that is shared with several other plant and relatives tend to occupy different macrohabitats and animal radiations. hence different communities (Elton 1946, Williams 1964, DIVERGENCE ORDER TEST Gotelli and Graves 1996, Cavender-Bares et al. 2004). The alternative, if a niche traits were more labile, would The divergence order test (DOT) was designed to facilitate coexistence and divergent resource use in address these questions of niche evolution, as well as sibling species within local communities. Divergence of other questions regarding the relative sequence of macrohabitat parameters among sibling taxa is compat- diversification events in a clade. The test examines the ible with the allopatric model of speciation, as disjunct relative timing of evolutionary divergence for two populations in a heterogeneous landscape are likely to continuous characters, and it is based on a modification encounter distinct habitats (Graham et al. 2004; but see of the method of phylogenetic independent contrasts Wiens [2004]). Differentiation of the a niche would then (Felsenstein 1985). Independent contrasts transform the represent a later stage of evolutionary divergence, either trait data for N species into a set of N – 1 contrasts, each resulting from or directly promoting species coexistence based on the difference between trait values across a as the ranges of the now distinct species expand into phylogenetic divergence. Under the assumption that the each other’s territory. trait evolved independently at each divergence, the In an important paper, Diamond (1986) argued for contrasts provide a robust basis on which to test this ‘‘habitat-first’’ model of speciation, based on his hypotheses of correlated evolution, addressing the observations that closely related bird species in New underlying historical pattern of trait evolution as well Guinea tend to be allopatric and occupy distinct as better meeting the assumptions of standard para- macrohabitats along elevational or climatic gradients metric statistics (Garland et al. 1992). For the DOT, we (see review by Schluter [2000]). In addition, Diamond modify this method and use the absolute differences and Schluter both argued that the habitat-first speci- between related nodes derived from maximum-like- ation model can be extended to the analysis of adaptive lihood estimates of ancestral trait reconstructions, radiations, based on a parsimonious assumption that obtained using ANCML (Schluter et al. 1997). This rates of evolution do not dramatically change. In other approach allows us to incorporate the uncertainty of words, if habitat divergence represents the first stage of reconstructions in deeper nodes. speciation among close relatives, then it would also be The divergence order test is based on two sets of characteristic of early speciation events at the base of an numbers: (1) the absolute value of the unstandardized adaptive radiation. As a corollary, if differentiation of a contrasts for trait i across the nodes (k ¼ 1, 2, ..., N)ofa niche occurs late in speciation, or is observed among phylogeny (Cik), which measures the magnitude of the distant relatives, then it would be characteristic of the divergence that occurred at each node regardless of the later stage of adaptive radiation. Streelman and Danley direction of change; and (2) the age of each node (Ak). (2003) presented a related model, arguing that vertebrate We then calculate a weighted mean age of divergence for adaptive radiations follow a trajectory of divergence each trait as follows: along three axes: habitat, trophic morphology, and XN communication, usually in that order. However, their AkCik use of ‘‘habitat’’ refers more to microhabitats within a k 1 W ¼ ¼ : ð1Þ community (e.g., benthic vs. limnetic sticklebacks), i XN rather than Diamond’s larger scale differentiation Cik among elevational bands and different forest types k ¼ 1 occupied by New Guinea birds. This ambiguity over The result is an average age, in units of time, that the use of the word habitat is unfortunate, as there is a indicates whether the large divergences in a trait tended substantive difference between these models in their to occur early or late in the diversification of a group emphasis on large-scale habitat differences, implying (Fig. 1). Note that this age will not generally correspond allopatric populations, vs. microhabitat differentiation to the age of any single divergence; it is simply a within local communities. statistical measure of the tendency toward early or late S52 D. D. ACKERLY ET AL. Ecology Special Issue

the absolute value of standardized independent contrasts and the standard deviation of those contrasts (the square root of subtending branch lengths) should be nonsignificant (Garland et al. 1992). A negative correlation would indicate larger contrasts than expected on rapid bifurcations, and vice versa for a positive correlation. The absolute values of standardized contrasts should also fit a half-normal distribution, and this can be checked visually using truncated normal probability plots. In addition, if several distinct clades are present (as in the Ceanothus case), homogeneity of evolutionary rates can be tested using a nonparametric comparison of standardized contrasts between groups (Garland 1992), or a recently introduced maximum-likelihood approach (O’Meara et al. 2005). In general, methods based on independent contrasts are fairly robust to violations of Brownian motion (Diaz-Uriarte and Garland 1996, Ackerly 2000), but this has not been evaluated for the calculation of standard errors by ANCML or the DOT analysis. It can be useful to examine correlations between the two traits under consideration, though DOT does not require that the traits exhibit any particular pattern of correlated or independent evolutionary change. If changes in the two traits are tightly linked, then DOT will certainly not be significant, as the contrasts will be similar in magnitude at each node. However, differences in the magnitude of a few basal or distal nodes could result in a significant DOT outcome, and trait evolution could still be correlated overall on the tree. FIG. 1. Example of the divergence order test (DOT). Two We have explored several approaches to significance patterns of trait divergence are illustrated on a simple phylogeny. Numbers at the tips of the phylogeny indicate two testing of the D statistic (see Appendix A). We present possible trait states, 0 or 1. Numbers at interior nodes (in italics) here our preferred method, based on a bootstrapping show the contrast for that node. Trait A (open circles) exhibits a approach, to obtain confidence intervals for the two pattern of late divergence, whereas Trait B (shaded circles) estimates of W and their difference, D. The rationale for exhibits a pattern of early divergence. The lower panel plots contrast magnitude vs. age and shows the calculated weighted this approach is that comparative methods, particularly divergence age for Trait A (WA ¼ 1 Ma [i.e., 1 million years independent contrasts, tend to underestimate the mag- ago]) and Trait B (WB ¼ 2 Ma). nitude of older divergences for rapidly evolving traits. As a simple example, consider a bifurcating tree with divergence for the trait in question. The DOT is then four species at the tips. If each pair of sister taxa has based on the comparison of the weighted divergence age divergent trait values, reflecting rapid trait evolution, for two traits (D ¼ Wi Wj) to determine whether one then the averages for their respective common ancestors trait diverged significantly earlier than the other, on could be virtually identical and the basal contrast will be average. nearly or exactly zero (e.g., Fig. 1, Trait A). However, The DOT statistic is derived from contrasts between given the rapid rate of evolution for this trait, it is also ancestral states and does depend on the accuracy of the possible that a large divergence occurred at the first ancestral estimates themselves (Oakley and Cunningham node, followed by reversals at the subsequent nodes 2000). We do not consider the traits of outgroups in resulting in convergence among extant taxa. Maximum- calculating ancestral states, as these are only necessary likelihood estimates of ancestral states allow for this to identify trends in trait evolution within a group, and possibility by placing confidence limits on the ancestors not the magnitude of divergences. The maximum- (Schluter et al. 1997). If a trait evolves rapidly, then the likelihood algorithm of ANCML assumes that the confidence limits at deeper nodes will be large (see pattern of trait evolution fits a model of Brownian Appendix A, Fig. A1). motion. Global squared-change parsimony, which is We use ANCML (Schluter et al. 1997) to generate also based on Brownian motion, provides the same maximum-likelihood estimates and confidence limits of ancestral estimates, but no confidence limits. The fit to ancestral states at each node; we then create bootstrap Brownian motion can be tested in several ways. First, distributions of the potential magnitude of each using Felsenstein’s (1985) algorithm, the correlation of divergence event (see Appendix A, Fig. A1). Hypo- July 2006 NICHE EVOLUTION AND ADAPTIVE RADIATION S53 thetical ancestral values are sampled from the distribu- Despite these consistent differences in drought toler- tion for each trait at each node, and from each sample ance and fire response, both clades are widespread in the we calculate the corresponding values of Cik, Cjk, Wi, California Floristic Province, inhabiting chaparral, Wj, and D. We then examine the distribution of D values semiarid forests, and oak woodland. Often, species pairs to determine whether D ¼ 0 falls outside the 95% representing one species from each of the subgroups co- confidence limits on the mean, indicating significance of occur, and it has been suggested that differences in fire the observed values at P 0.05. The calculation and response and/or tolerance of water stress facilitate this significance of the DOT method were implemented on coexistence (Keeley 1977, Keeley and Zedler 1978, Davis Mac OS X, using awk scripts, R (R-project 2004), and et al. 1999). These patterns suggest that the basal ANCML (see Supplement). divergence between the two clades involved a niche traits related to drought tolerance or postfire regeneration Branch lengths and node ages strategies. In contrast, both clades are represented by As the objective of this test is to calculate relative species throughout California, suggesting more recent timing of divergence events, the analysis should ideally differentiation of the climatic niche envelope, represent- be conducted with ultrametric, calibrated phylogenies ing the b niche (Knight and Ackerly 2001). Here we based on a molecular clock, or rate-smoothed branches undertake four new analyses to quantify and test these if the branch lengths violate a molecular clock (Sander- observations. (1) We have assembled a co-occurrence son 2002). Methods for obtaining relative ages are data set from the literature, and we use null models to improving, although there can still be considerable test for nonrandom patterns of co-occurrence between uncertainty due to heterogeneous rates of molecular species from the two subgroups. (2) We combine the coevolution and difficulty in establishing calibration points occurrence data with a trait data set to test whether co- across different clades (Sanderson 2002, 2003). The occurring species differ significantly for traits related to DOT method will be robust to much of this uncertainty, plant growth strategies (a niche) and are more similar because the same ages are used in the calculation of than expected for traits related to climatic envelopes (b weighted divergence times for both traits. For the nodes niche). (3) We reanalyze the Ceanothus phylogeny, based along any contiguous path from the root to the tips, ages on internal transcribed spacer (ITS) sequence data, to will always be correctly ordered, even if the actual values obtain an ultrametric tree with branch lengths fit to a are uncertain. Problems would most likely arise if molecular clock. (4) We use this phylogeny and the trait incorrect calibrations were applied to two or more data set to apply the DOT analysis, testing the independent segments of the tree (i.e., along different prediction that a niche traits diverged earlier than b root-to-tip paths) in which different traits exhibited niche traits in the evolution of Ceanothus. large evolutionary divergences. Attention to this prob- METHODS lem is warranted in applications of the test. Occurrence data CASE STUDY:COMMUNITY ASSEMBLY AND NICHE A matrix of co-occurrence data for Ceanothus in EVOLUTION IN CEANOTHUS California was obtained from a search of the literature The woody plant group Ceanothus comprises ;55 and consultation with colleagues. A total of 51 sites were minimum-rank taxa (species or subspecies) that have obtained that had two or more co-occurring Ceanothus, primarily diversified within the California Floristic with a total of 16 different taxa (plots in the same Province (McMinn 1942, Hardig et al. 2000). The group location with the same species composition were is divided into two well-supported clades (Jeong et al. recorded as one site; Nicholson 1993). Of these sites, 1997, Hardig et al. 2000) that differ consistently in 35, with 13 taxa, were located in chaparral of the Coast several morphological and physiological traits related to or Transverse ranges, while 16 sites with 7 taxa were drought tolerance. The two groups are considered from the Sierra Nevada region (CT and SN, respec- subgenera, and are currently designated Cerastes and tively). Of the 51 sites, 48 had just two Ceanothus Ceanothus; for greater clarity (and consistency with species, while the remainder had three. (See Appendix B phylogenetic naming conventions), we prefer the older for details of the occurrence data matrix.) name Euceanothus for the latter group. Species in Cerastes have thick leaves with stomatal crypts and have Trait selection shallower roots and more embolism-resistant xylem than Although coexistence of Ceanothus species, in a matrix do members of Euceanothus (McMinn 1942, Hellmers et of other taxa in the community, can involve contrasting al. 1955, Davis et al. 1999). Additionally, species of physiological or regeneration strategies, no direct studies Cerastes establish only from seed following fire, while of coexistence mechanisms in chaparral have been Euceanothus species generally resprout as well (Wells conducted. To reflect differences in drought tolerance, 1969, Schwilk and Ackerly 2005). In a Mediterranean- the ideal traits would be either a direct measure of xylem type climate, seedlings of nonsprouting species must tolerance to embolism under water deficit or wood survive an intense summer drought period after winter or density, which is a close correlate of xylem tolerance spring germination. (Hacke et al. 2001). These traits are known to vary S54 D. D. ACKERLY ET AL. Ecology Special Issue between the two subgroups (Davis et al. 1999), but they niche parameters show greater similarity among co- are not available for large numbers of species. As a proxy occurring species than expected. The measure of trait for contrasting physiological strategies, we used specific dissimilarity within communities was simply the differ- leaf area (SLA), the ratio of fresh leaf area to dry mass. ence between species values for two-species samples, and This well-studied leaf functional trait plays an important the mean of the successive differences among ranked role in the ‘‘leaf economic spectrum’’ of variation in plant values in three-species samples. We also calculated the metabolic rates (Wright et al. 2004). In general, species mean trait value for each community, which allowed us with higher SLA have shorter leaf life span and higher to test our additional null models. (4) The standard photosynthetic rates. In chaparral shrubs such as deviation of site means should be higher for climate niche Ceanothus, high SLA is associated with less drought- parameters than expected by chance, due to turnover of tolerant leaves (Ackerly 2004b), and we have a large data species along climatic gradients. Finally, (5) among-site set available for roughly two-thirds of the Ceanothus taxa standard deviation in mean SLA should be lower than (Ackerly 2004a). We do not claim that differences in SLA expected under the null, as the combinations of species per se promote coexistence, but rather that they could be from the two clades result in high trait disparity within associated with suites of traits that are related to niche sites and low disparity across sites. One-tailed tests were partitioning and differentiation in ecological strategies of conducted for all hypotheses, based on these predictions, co-occurring species. Data for SLA are species means comparing the observed data to 999 randomizations. collected previously from field, herbarium, and botanic As a null model, we use the ‘‘independent-swap’’ garden specimens (Ackerly 2004a); three new taxa that algorithm (Gotelli and Entsminger 2001), preserving appeared in the occurrence data set were added to this both site diversity and species frequency of occurrence trait data set (C. greggii, C. parvifolius, and C. sanguineus) while randomizing assignments of species to sites. This is based on measurements of specimens in the University critical to ensure that patterns of trait assembly do not and Jepson Herbaria (University of California–Berkeley, simply reflect differential abundance of particular California, USA). Values were log-transformed for all species. All calculations were carried out in R (R-project analyses, as relative differences are a better measure of 2004); the swap algorithm was implemented by S. physiological differentiation (Reich et al. 1997). (See Kembel in C as part of the PHYLOCOM package Appendix C for details of the trait data set.) (Webb et al. 2004). At larger spatial scales (b niche), Ceanothus are differentially distributed with respect to edaphic con- Phylogeny ditions (e.g., serpentine specialists), habitat (chaparral The Ceanothus phylogeny of Hardig et al. (2000) was vs. conifer forests), elevational range, and macroclimate reanalyzed to obtain an ultrametric tree fit to a (precipitation and temperature) (Hickman 1993, Nich- molecular clock for the taxon sample in our trait data olson 1993, Davis et al. 1999). We quantified the realized set. Conflicting phylogenies for Ceanothus based on ITS climatic niche, to characterize these large-scale distribu- vs. matK sequence data could reflect lineage sorting tions, by overlaying species distributions on climate during rapid radiations or hybridization (Hardig et al. maps of California and calculating mean climatic 2000), and ITS (which is a nuclear marker) was selected parameters for each species (Knight and Ackerly as a more reliable estimator of the ‘‘true’’ species tree. 2001). We have selected the mean precipitation and the Limited sampling of ndhF (10 taxa; Jeong et al. 1997) is mean January temperatures within the geographic range insufficient to incorporate in the broader analysis of each species, reflecting distributions along geographic considered here. ITS sequence data for 76 accessions and elevational gradients in California. The climate (73 Ceanothus and 3 outgroups [Adolphia californica, nicheanalysisservesasanindirectsurrogatefor Zizyphus obtusifolia, and Spyridium parvifolium]) were unmeasured physiological traits related to species obtained from GenBank (accessions GBAN-AF048901 tolerances and distributions along climate gradients. through GBAN-AF048975; Hardig et al. 2000). Sequen- This interpretation assumes that distributions do not ces were aligned with ClustalX using default parameters, simply reflect historical factors and limited dispersal and alignments were checked by eye (no manual potential. The contraction and expansion of chaparral adjustments were made); total aligned sequence length during the interglacial periods (Graham 1999) argues for ITS1, ITS2, and the intervening 15S region was 627 against a strong role for dispersal limitation as a long- nucleotides. Taxa with identical sequences were kept in term constraint on species distributions. the analysis, for use later in comparative analyses. Multiple sequences for individual taxa were pruned to Community assembly one representative sequence, based on preliminary We used five null models for community assembly to analyses (Hardig et al. 2000). The resulting analysis test several predictions, relative to patterns that would be included 56 Ceanothus sequences and 96 informative expected by chance: (1) Species from the two clades characters. Phylogenetic analysis was conducted with (Cerastes and Euceanothus) co-occur more often than PAUP*, using parsimony criteria and a heuristic search expected. (2) Values of SLA show greater variation (random addition sequence with 10 replicates, TBR, among co-occurring species than expected. (3) Climate MULTREES in effect, collapse zero-length branches in July 2006 NICHE EVOLUTION AND ADAPTIVE RADIATION S55 effect, and steepest descent not in effect) (Swofford polytomy). Zero-length branches create problems for 2002). This analysis resulted in 174 equally parsimo- both ANCML and independent contrasts, as they imply nious trees of length 322 on one island, which was hit in instantaneous evolutionary divergence (a hard polyto- all 10 replicate searches. The strict consensus of the my), whereas they might reflect uncertainty of the equally parsimonious trees was very similar to the results sequence of speciation events (soft polytomy) as much reported by Hardig et al. (2000). The most significant as rapid radiation. Zero-length branches represent difference was that we obtained more resolution within truncated estimates of elapsed time since speciation, Euceanothus, with the Western group (Hardig et al. since each branch will remain at zero length until the first 2000: Fig. 1) monophyletic and nested within a para- fixed base change occurs. We addressed this problem by phyletic Eastern group. adjusting zero-length branches to a small nonzero value For comparative analysis, ANCML (Schluter et al. (1.0 3 104) slightly lower than the shortest branches 1997) requires fully bifurcating phylogenies. It is resulting from the maximum-likelihood fit to the possible to generate these by randomly resolving the molecular clock, which ranged from 1.03 3 104 to 4.94 trees obtained in the analysis just described (in which 3104 across the 100 trees. Based on our calibration, this zero-length branches were collapsed), but we are not adjustment represents an absolute time of ;5 3 104 yr. aware of software that will provide alternative reso- Note that these small divergences (especially if they occur lutions while maintaining branch lengths. As an on both sides of a bifurcation) will lead to an inflated alternative, we conducted a second parsimony search estimate of the Brownian motion rate parameter and, in PAUP* with the same parameters, but with zero- hence, broader confidence intervals on ancestral states. length branches not collapsed and MAXTREES ¼ This will increase the standard errors of weighted 10 000. Ceanothus oliganthus ssp. oliganthus, which was divergence age (W ) from our bootstrap procedure, present in our ecological data but missing from the leading to a more conservative test of significance for molecular data, was added to the matrix with the same the DOT statistic. We recognize that this is an imperfect sequence as C. oliganthus ssp. sorediatus. Given taxa solution and hope that the problem of zero-length with identical sequences, this search quickly reached branches will be addressed in future research on branch the maximum number of trees, with length ¼ 322, as in length calibration for comparative analysis. the prior case. We then pruned the trees to include only Divergence order test analysis the 39 taxa for which we had phenotypic data, resulting in 3254 unique topologies (outgroups were not We conducted the DOT analysis based on the average considered in the analysis of ancestral states). For age of internal nodes, weighted by the absolute value of further analysis, 100 trees were selected from this set by the unstandardized independent contrasts for each trait. sampling every 20th tree (because immediately adjacent Significance of the difference in ages was assessed by trees tend to be similar to each other). This set of 100 bootstrapping trait histories from the mean and stan- alternative, fully bifurcating trees was used for all dard deviations obtained from ANCML. Given the subsequent analyses. strong preliminary data that we have introduced, we conducted one-tailed tests of the hypothesis that SLA Branch lengths divergence occurred earlier than divergences in climate Maximum-likelihood methods were used to fit branch niche parameters. lengths to the 100 topologies, based on the HKY85 RESULTS model with transition/transversion rates fit empirically from the data (Swofford 2002). A molecular clock was Trait variation not rejected using this model (P . 0.05 for all trees), so Across the entire trait data set (39 taxa), specific leaf the branch lengths fit with a molecular clock were used area (SLA) was significantly higher in Euceanothus than for comparative analyses. Based on an independent Cerastes. Precipitation and SLA were weakly correlated analysis of rates of rbcL evolution in Ceanothus (Jeong overall (R ¼ 0.25), and they were essentially independent et al. 1997), the split between Cerastes and Euceanothus based on independent contrasts (R ¼ 0.04) (Fig. 2A). was calibrated at 18–39 Ma. Fossil evidence provides January temperature and SLA were negatively corre- independent confirmation of the minimum age, as taxa lated across species (R ¼0.35) and based on assignable to both clades appear in the fossil record by independent contrasts (R ¼0.31). This negative 18 Ma (Chaney 1927, Axelrod 1956). Using this relationship was also apparent within Euceanothus, but calibration for the basal node, the clock-calibrated tree not in Cerastes (Fig. 2B). The decline in Euceanothus suggests that radiation of each of the two (Cerastes and reflects the transition from deciduous taxa occupying the Euceanothus) began no more recently than 4–5 Ma, at coldest ranges (e.g., C. parvifolius) to evergreens at lower about the same time as the onset of the Mediterranean- latitudes or elevations. The most striking aspect of both type climate in California. relationships is the marked difference in SLA between The inclusion of taxa with identical sequences (a the clades that is maintained across the climatic common occurrence for rapidly speciating groups) gradients, consistent with the prediction of local co- results in zero-length branches in the phylogeny (i.e., a occurrence between species that differ in SLA. S56 D. D. ACKERLY ET AL. Ecology Special Issue

2 FIG. 2. Speciﬁc leaf area (SLA; originally measured in mm /mg) vs. (A) mean precipitation, and (B) January temperatures within species ranges for species of Cerastes (solid circles) and Euceanothus (open circles).

Community assembly perhaps due to distribution of evergreen vs. deciduous Taxonomic co-occurrence.—Of the 48 sites with two (low- vs. high-SLA) species along elevational gradients. species, 38 had one from each of the two major clades As expected, climate niche parameters are always (Cerastes and Euceanothus); this pattern was particularly similar among co-occurring species and significantly striking in the Coast/Transverse (CT) chaparral sites (31 different among sites. of 35 sites), but was not significant in the Sierra Nevada Divergence order test analysis (SN) (Table 1). For both the full data set and the coastal partition, the elevated co-occurrence of species from These results support the selection of SLA and different clades was highly significant, relative to the null realized climate niche parameters as traits reflecting model that maintained both site diversity and species local (a) vs. regional (b) differentiation, respectively. The occurrence (P 0.001). The frequent co-occurrence of patterns are stronger in the Coast and Transverse taxa from the two clades has long been noted in the ranges, and if there were a monophyletic group in literature, but not previously quantified and tested Ceanothus restricted to these communities we would relative to a null model. limit our analyses to this group. However, the evolu- Trait disparity.—Across all sites, the mean difference tionary radiation into the two major clades, and in log10(SLA) between co-occurring species was 0.32 subsequently within clades, encompasses both geo- units, and as predicted this value was significantly graphic areas (and beyond). We feel it is important to greater than the null expectation (P , 0.04; Table 2). In contrast, differences between climate niche parameters of co-occurring species were much smaller than expected TABLE 1. Relative frequency of local co-occurrence patterns (P 0.001 in both cases; Table 2). When the data are for Ceanothus, in terms of the number of species from each major subgroup (CT ¼ Coast and Transverse ranges; SN ¼ partitioned geographically, the within-site disparity in Sierra Nevada). SLA was still significantly greater in CT, but disparity in SN was less than expected. Disparity in climate niche No. communities Community pattern parameters is significantly lower than the null, as Total CT SN predicted, in both areas (Table 2). The among-site Cerastes Euceanothus (P 0.001) (P 0.001) (NS) standard deviation in trait means was significantly 02 7 25 greater than expected for climate niche traits, as 1 1 38 31 7 predicted, in the entire data set and both geographic 20 3 21 partitions. For SLA, it was significantly lower than 03 2 02 12 1 01 expected in CT, but the pattern was reversed in SN 21 0 00 (Table 2). 30 0 00 Collectively, these analyses indicate that in Coast and Notes: Example of how to read Table 1: The first row Transverse range chaparral, co-occurring Ceanothus indicates that a total of seven communities were recorded (two species exhibit greater disparity in SLA than expected in CT and five in SN) with two co-occurring Euceanothus under a null model of community assembly. In species and no Cerastes. P values indicate the probability of obtaining the observed number of sites occupied by species contrast, in the Sierra Nevada it appears that SLA from the two subgroups, relative to a null model of community varies among sites, while within-site disparity is low, assembly (see text). July 2006 NICHE EVOLUTION AND ADAPTIVE RADIATION S57

TABLE 2. Mean trait disparity among co-occurring species, and standard deviation of trait means among sites.

Mean disparity SD of site means Trait Prediction All (N ¼ 51) CT (N ¼ 35) SN (N ¼ 16) Prediction All (N ¼ 51) CT (N ¼ 35) SN (N ¼ 16) Specific leaf area (mm2/mg, log) Observed mean . 0.32 0.35 0.25 , 0.158 0.093 0.170 Expected mean (SD) 0.29 (0.018) 0.28 (0.021) 0.28 (0.016) 0.17 (0.012) 0.15 (0.013) 0.14 (0.014) P ,0.04 0.001 NS NS 0.001 NS Annual precipitation (mm) Observed mean , 117 123 103 . 217 159 83 Expected mean (SD) 280 (25.2) 187 (17.3) 121 (8.7) 155 (12.1) 124 (10.5) 67 (8.22) P 0.001 0.002 ,0.03 0.001 0.002 ,0.02 January minimum temperature (8C) Observed mean , 1.25 0.92 1.95 . 2.001 0.985 1.571 Expected mean (SD) 2.53 (0.19) 1.24 (0.093) 2.49 (0.21) 1.50 (0.11) 0.81 (0.065) 1.23 (0.14) P 0.001 0.002 , 0.02 0.001 , 0.01 ,0.02 Notes: Observed values are compared to expectations based on a null model of community assembly. Analyses were conducted for all sites, and for Coast and Transverse ranges (CT) and Sierra Nevada (SN) sites separately. Prior predictions regarding the direction of the difference between observed and expected values are listed, and all significance tests are one-tailed. conduct evolutionary analyses on all available data for For all 100 alternative phylogenies, weighted divergence the entire group. ages were older for SLA than for both climate The three traits considered here generally fit the parameters, and the DOT was significant in 94 of 100 Brownian motion model underlying the use of ANCML trees for the SLA vs. January temperature difference, for ancestral states. For all three traits, inspection of and for 92 out of 100 trees for SLA vs. precipitation. normal probability plots for the absolute value of DISCUSSION standardized contrasts indicated a close fit to a truncated normal distribution. For SLA and January This analysis of the radiation of Ceanothus demon- temperatures, correlations of standardized contrasts and strates an initial shift in a niche traits that subsequently their standard deviation (i.e., the square root of the promote (or at least facilitate) coexistence among related subtending branch lengths) were not significant. For species. These traits were then conserved as the radiation precipitation, the correlation was significantly negative progressed, and later speciation events were character- across all trees, reflecting larger than expected divergen- ized primarily by divergence in climate envelopes, ces across rapid speciation events, and a small diver- corresponding to geographic differentiation along lat- gence across the long branches between Cerastes and itudinal and elevational gradients. The result is that co- Euceanothus. This pattern will lead to a relatively high occurring Ceanothus species within local communities Brownian motion rate parameter, to accommodate these are more distantly related than expected by chance, relative to the group as a whole. rapid divergences, and will thus inflate the confidence Similar patterns are evident in other clades that have intervals around the ancestral estimates for precipita- been analyzed in a phylogenetic context. In the oaks tion, making the DOT more conservative. Across most (Quercus) of Florida, local communities tend to be of the 100 trees, rates of trait evolution were homoge- phylogenetically overdispersed, often with one or two neous between Cerastes and Euceanothus; in 3 and 24 members each drawn from three distinct clades (red, trees, rates of SLA and January temperature evolution, white, and live oaks). Habitat preferences diverge respectively, were significantly higher in Euceanothus repeatedly within each of these clades; deeper divergen- (0.04 , P , 0.05). ces between the clades involve traits, such as seed Fig. 3 illustrates trait divergences on a randomly maturation time and wood density, which may promote selected tree from the analysis. The largest contrast for coexistence through differential regeneration or patho- SLA was located at the basal node between Cerastes and gen tolerance, respectively (Cavender-Bares et al. 2004). Euceanothus (Fig. 3). The weighted divergence age for In studies of Phylloscopus warblers in Kashmir, Rich- SLA, averaged across the 100 alternative phylogenies, man and Price (1992, Richman 1996) argued that deep 3 was 6.5 3 10 branch length units (Table 3). For the divergences in the group involved differentiation in climate parameters, the basal contrasts were much feeding strategies, while more recent speciation events smaller, and larger divergences were noted within each were related to macrohabitat distributions (coniferous of the two major clades (Fig. 3). The weighted vs. deciduous forests) (but see Forstmeier et al. [2001]). divergence ages were 4.30 3 103 and 4.24 3 103 for In the radiation of Anolis lizards, distinctive ecomorphs, January temperatures and precipitation, respectively. which coexist by feeding in different parts of the canopy, S58 D. D. ACKERLY ET AL. Ecology Special Issue

groups (Glor et al. 2003). In our terminology, the earlier divergence events in each of these cases involve shifts in the a niche, whereas b niche traits continue to diverge later in the radiation. Streelman and Danley (2003) include the anoles as one of the case studies in their review, considering the diversification of ecomorphs as an example of the first stage of radiation, involving microhabitat divergence. This is consistent with our interpretation of ecomorphs as diversification of the a niche, although our terminology is different. These case studies provide a striking contrast to Diamond’s (1986) habitat-first model, which proposed that the first stage of speciation and adaptive radiation involved allopatric divergence along habitat gradients. These contrasting interpretations reflect differences in the interpretation of fast- vs. slow-evolving traits, and they highlight underlying philosophical differences in the interpretation of comparative data. It is well known that phylogenetic inference works best for slowly evolving traits (Felsenstein 1988). Rapid evolution erodes the signal of early events on a phylogeny, due to reversals on the same or adjacent branches and convergence among the terminal states (strictly speaking, this is only true for traits with a finite number or range of states; Donoghue and Ree 2000, Ackerly and Nyffeler 2004). For this reason, independent contrasts will generally provide an extremely poor estimate of the timing of divergence for rapidly evolving traits, as it will appear that there was little divergence in deeper nodes (e.g., Fig. 1). The high level of uncertainty in ML ancestral state reconstructions reflects this problem for rapidly evolving traits (Schluter et al. 1997, Cunningham et al. 1998) and led us to adopt the bootstrap method proposed here. In this situation, if the most recent speciation events in an adaptive radiation involve divergence in macrohabitat or habitat-related traits, it is parsimonious to assume that deeper events also involved such divergences (T. Price, personal communication), though evidence of this will not be available from phylogenetic analysis. FIG. 3. Divergence order test (DOT) for Ceanothus, In contrast, for slowly evolving traits phylogenetic illustrated for one randomly chosen tree out of 100 equally methods are quite powerful, leading to a different parsimonious trees used for analysis. For all panels, ages along interpretation of the comparative data. For this case, the x-axis are clock-calibrated molecular branch length units; the breaks in the axis indicate the long branches connecting the as in the contrasting SLA values in Ceanothus,or two recently diversifying clades. (A) Ceanothus phylogeny, divergent ecomorphs in Anolis, the greatest trait differ- based on reanalysis of internal transcribed spacer (ITS) ences will generally be observed among distantly related sequence data from Hardig et al. (2000). Species names are species. The conservatism of these traits in diverse clades omitted for clarity. (B–D) Open circles indicate the magnitude within each group strongly argues that the underlying of unstandardized contrasts vs. node age for specific leaf area (SLA), January temperatures, and mean precipitation, respec- divergences occurred during the initial speciation events tively (see Table 3 for weighted mean ages). Standard errors of early in the overall radiation. The view that a niche the contrasts (derived from the bootstrap procedure) are divergence occurs late in the course of species differ- illustrated for the basal contrast only. The solid diamonds entiation, because these traits tend to differ between indicate weighted mean divergence age for each trait (6SE) distantly related species, is not compatible with com- based on 200 bootstrap randomizations of the contrasts (vertical position of this point is arbitrary). parative phylogenetic analysis. Taken together, these views lead to a possible have evolved independently on multiple islands (Losos synthesis of contrasting interpretations. In the cases et al. 1998, Knouft et al. 2006). On the larger islands, discussed here, adaptive radiation may have proceeded however, there has been continued speciation involving by ‘‘a niche early’’ and ‘‘b niche throughout.’’ In other diversification of macrohabitats within ecomorph words, habitat divergence can occur frequently at all July 2006 NICHE EVOLUTION AND ADAPTIVE RADIATION S59

TABLE 3. Results of the divergence order test (DOT) for relative divergence times of speciﬁc leaf area (SLA) and climate niche parameters in Ceanothus.

Trait Mean age (SE) D (SE) No. nonzero SLA 0.00654 (0.00083) January temperature 0.00430 (0.00071) 0.00225 (0.00108) 94 Precipitation 0.00424 (0.00083) 0.00230 (0.00117) 92 Notes: Mean age is the average, over 100 alternative trees, of the weighted divergence times (W) for each trait. For each tree, W is derived by bootstrapping the ancestral states from maximum likelihood distributions for ancestral states, with 200 replicates; D is the mean difference in age of each climate parameter vs. SLA, over the 100 alternative trees; no. nonzero is the number of trees (out of 100) in which D was significantly different from zero, based on a one-tailed test of the hypothesis that mean age for SLA was older. depths in the tree, although it can only be reconstructed generate stabilizing selection effects that lead to evolu- with confidence in recent speciation events. Habitat tionary stasis in niche parameters (Holt 1987, 2003, differences clearly can play an important role in Kirkpatrick and Barton 1997, Case and Taper 2000). allopatric speciation, though it is important to note that Empirical studies of these predictions are needed. The allopatric populations might also occupy similar envi- roles of niche conservatism in speciation, the evolution of ronments in different geographic areas (Peterson et al. regional biota and the assembly of communities has 1999, Wiens 2004). In some cases, speciation also recently received increased attention (Webb et al. 2002, involves a shift in a niche traits related to resource Ackerly 2003, Wiens 2004), and each of these offers a partitioning in local communities, but these events are counterpoint to the emphasis on ecological divergence as apparently less frequent. Divergence in a niche traits a key component of evolutionary radiations. might be due to incidental divergence under conditions The conclusion that a niche traits evolve relatively that favor different traits (e.g., island vs. mainland slowly during an adaptive radiation contrasts with the populations), or divergence by character displacement in views of Silvertown et al. (2006a, b) on niche evolution. sympatry due to direct competitive interactions between They argue, based on several lines of evidence, that the a the incipient species (Schluter 2000, Levin 2004). niche evolves rapidly, and as a corollary local commun- If correct, the ‘‘a early, b throughout’’ model presents ities usually show little phylogenetic signal in their two unresolved questions about the evolution of a niche species composition. This conclusion is supported by traits. First, why should these traits exhibit evolutionary their analysis of the phylogenetic structure of English transitions early on in adaptive radiations? (At the time meadow communities (Silvertown et al. 2001, 2006a, b) of this initial divergence, the adaptive radiation is still an and by comparison of niche overlap between congeneric unrealized future of the clade.) And second, why do a and noncongeneric species within local communities. niche traits often exhibit phylogenetic conservatism The apparent conflict between their analyses and our during subsequent diversification. With regard to the conclusions is most likely due to the different scales of first question, it is tempting to identify divergence in a analysis and sampling of taxa. The species of the English niche traits as key innovations, or invasions of a novel meadow communities are widely dispersed across the adaptive zone (sensu Simpson 1953), contributing to the angiosperm phylogeny. When the phylogeny is pruned subsequent radiation. For example, in the case of for analysis of niche distributions, the closest relatives Ceanothus we have strong evidence that California remaining on the tree are rarely if ever immediate chaparral communities are capable of supporting at sibling species. As a result, even the most recent ‘‘events’’ least one sprouting and one nonsprouting Ceanothus representedonsuchaphylogenyarerelativelyold species. Thus, when this trait diverged early in the compared to our analysis of adaptive radiations. Thus, evolution of Ceanothus (the direction of evolution Silvertown et al.’s (2006a) conclusions and our analyses between ancestral and derived states is unknown), the may be entirely compatible, but focused on different two descendent subclades were both able to diversify in scales of analysis. parallel across a broad gradient of climatic conditions. While such scenarios are plausible, and may be correct, CONCLUSIONS it is difficult to conduct rigorous analyses of diversifi- The divergence order test (DOT) introduced here cation hypotheses in terms of the timing of phenotypic provides a quantitative approach to test hypotheses innovation and hypothesized shifts in speciation rates about the relative sequence of divergence for continuous (Sanderson and Donoghue 1994, Hodges 1995). traits. Considering the potential pitfalls in comparative The second question addresses the important topic of analysis of rapidly evolving traits, the bootstrap method niche conservatism: What are the mechanisms promoting incorporating the uncertainty of ancestral state recon- evolutionary stasis in ecological traits through speciation structions provides a conservative approach for signifi- and diversification of a clade? Recent theoretical analyses cance testing. Our application of the DOT to Ceanothus, support the view that contrasting selection pressures in and interpretation of other cases in the literature, leads heterogeneous environments, combined with gene flow, to the conclusion that a niche traits often diverge early interspecific competition, and/or habitat selection, can in the course of adaptive radiations. The b niche traits, S60 D. D. ACKERLY ET AL. Ecology Special Issue which are related to macrohabitat distributions, might Elton, C. 1946. Competition and the structure of ecological evolve rapidly throughout the radiation, although the communities. Journal of Animal Ecology 15:54–68. Felsenstein, J. 1985. Phylogenies and the comparative method. signal of early divergences is erased by the high rates of American Naturalist 125:1–15. evolution. Further application of the DOT, or improved Felsenstein, J. 1988. Phylogenies and quantitative characters. tests addressing these questions, will provide an impor- Annual Review of Ecology and Systematics. 19:445–471. tant step towards synthesis of niche evolution and Forstmeier, W., O. Bourski, and B. Leisler. 2001. Habitat choice in Phylloscopus warblers: the role of morphology, adaptive radiation. phylogeny and competition. Oecologia 128:566–576. Garland, T., Jr 1992. Rate tests for phenotypic evolution using ACKNOWLEDGMENTS phylogenetically independent contrasts. American Naturalist D. D. Ackerly thanks C. O. Webb and J. B. Losos for the 140:509–519. invitation to contribute this paper to the ESA symposium and Garland, T., Jr., P. H. Harvey, and A. R. Ives. 1992. Procedures this special issue on Phylogenetics and Community Ecology. for the analysis of comparative data using phylogenetically The authors thank D. Schluter and T. Price for valuable independent contrasts. 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APPENDIX A Discussion of alternative null models (Ecological Archives E087-110-A1).

APPENDIX B Ceanothus occurrence matrix (Ecological Archives E087-110-A2).

APPENDIX C Ceanothus trait matrix (Ecological Archives E087-110-A3).

SUPPLEMENT Source code for DOT (Ecological Archives E087-110-S1). Ecology, 87(7) Supplement, 2006, pp. S62–S75 Ó 2006 by the Ecological Society of America

PHYLOGENETIC DISPERSION OF HOST USE IN A TROPICAL INSECT HERBIVORE COMMUNITY

1,6 2,7 3 4 5 GEORGE D. WEIBLEN, CAMPBELL O. WEBB, VOJTECH NOVOTNY, YVES BASSET, AND SCOTT E. MILLER 1Department of Plant Biology, University of Minnesota, Saint Paul, Minnesota 55108 USA 2Section of Evolution and Ecology, University of California, Davis, California 95616 USA 3Institute of Entomology, Czech Academy of Sciences and Biological Faculty, University of South Bohemia, 370 05 Ceske Budejovice, Czech Republic 4Smithsonian Tropical Research Institute, Balboa, Ancon, Panama 5National Museum of Natural History, Smithsonian Institution, Washington, D.C. 20013-7012 USA

Abstract. Theory has long predicted that insect community structure should be related to host plant phylogeny. We examined the distribution of insect herbivore associations with respect to host plant phylogeny for caterpillars (Lepidoptera), beetles (Coleoptera), and grasshoppers and relatives (orthopteroids) in a New Guinea rain forest. We collected herbivores from three lineages of closely related woody plants and from more distantly related plant lineages in the same locality to examine the phylogenetic scale at which host specificity can be detected in a community sample. By grafting molecular phylogenies inferred from three different genes into a supertree, we developed a phylogenetic hypothesis for the host community. Feeding experiments were performed on more than 100 000 live insects collected from the 62 host species. We examined patterns of host use with respect to the host plant phylogeny. As predicted, we found a negative relationship between faunal similarity, defined as the proportion of all herbivores feeding on two hosts that are shared between the hosts, and the phylogenetic distance between hosts based on DNA sequence divergence. Host phylogenetic distance explained a significant fraction of the variance (25%) in herbivore community similarity, in spite of the many ecological factors that probably influence feeding patterns. Herbivore community similarity among congeneric hosts was high (50% on average) compared to overlap among host families (20–30% on average). We confirmed this pattern using the nearest taxon index (NTI) and net relatedness index (NRI) to quantify the extent of phylogenetic clustering in particular herbivore associations and to test whether patterns are significantly different from chance expectations. We found that 40% of caterpillar species showed significant phylogenetic clustering with respect to host plant associations, somewhat more so than for beetles or orthopteroids. We interpret this as evidence that a substantial fraction of tropical forest insect herbivores are clade specialists. Key words: community ecology; community phylogenetics; herbivory; host specialization; host specificity; plant–insect interactions; phylogenetic dispersion; phylogeny; tropical rain forest.

INTRODUCTION insects have tended to focus on the reconstruction of In the era before automated DNA sequencing and ancestral associations for particular groups (Kelley and Farrell 1998) or whether particular insect groups and molecular phylogenetics, Daniel H. Janzen stated that their host plants have diversiﬁed in parallel (Farrell and ‘‘the systematics and taxonomy of interactions is hope- Mitter 1990, 1998). Other macroevolutionary studies less’’ (Janzen 1977). As robust phylogeny estimates for have examined patterns of phylogenetic conservatism in plants and insects become available, investigating the the host plant associations of phytophagous insects evolutionary history of their interactions is no longer a (Farrell 1998, Janz and Nylin 1998, Ward et al. 2003). fruitless endeavor. It is now possible to examine the Ecologists interested in patterns of herbivore com- historical associations of plants and insects by compar- munity structure are faced with a different set of ing molecular phylogenies for the interacting lineages questions. For example, to what extent do insects feed (Becerra 1997, Weiblen and Bush 2002, Percy et al. on closely related host plants in a particular community? 2004). Phylogenetic studies of host use by phytophagous How likely are host shifts to occur between divergent host lineages? Few studies have attempted to integrate Manuscript received 24 January 2005; revised 20 June 2005; the knowledge of phylogeny in the study of community accepted 23 June 2005. Corresponding Editor: A. A. Agrawal. structure (Connor et al. 1980, Strong et al. 1984, For reprints of this Special Issue, see footnote 1, p. S1. Marquis 1991, Losos 1996, Ødegaard 2003, Ødegaard 6 E-mail: [email protected] 7 Present afﬁliation: Arnold Arboretum of Harvard Uni- et al. 2005). Concern over the lack of statistical versity. independence among species led Kelly and Southwood S62 July 2006 PHYLOGENY OF HOST ASSOCIATIONS S63

(1999) to control for phylogenetic effects in demonstrat- integrated across three genes and rate-smoothed across ing that host plant abundance can predict herbivore the community phylogeny using penalized likelihood. If species richness in the temperate forest of Britain. But herbivores tend to feed on closely related plants more still more can be learned from phylogeny. The members than on distantly related plants, as we expect, then of any biotic community are related in some fashion, faunal similarity should decline with increasing phylo- and insights can be gained by examining ecological genetic distance between host species. patterns with respect to patterns of descent from Indices of phylogenetic dispersion that incorporate common ancestors. null models can be especially useful as quantitative tests The incorporation of phylogenetic knowledge in of host specificity in community samples. We used the ecological studies can inform our understanding of nearest taxon index (NTI) and net relatedness index community structure (Webb et al. 2002) and of evolu- (NRI) to quantify the extent of phylogenetic clustering tionary constraints on the distribution of traits in in particular herbivore associations and to test whether ecological communities (Chazdon et al. 2003). A useful patterns are significantly different from chance expect- approach is to apply clustering indices to the phyloge- ations (Webb 2000, Webb et al. 2002). These indices netic distribution of species that belong to a particular measure the mean phylogenetic distance between plants community sample drawn from a larger species pool that share a particular herbivore, relative to the mean (Futuyma and Gould 1979, Webb 2000). Such indices and standard deviation of herbivore associations ran- were first applied to the distribution of phytophagous domly distributed on the phylogeny, as obtained by insects across a host plant phylogeny in order to quantify multiple iteration. The NRI measures the average diet breadth (Symons and Beccaloni 1999, Beccaloni and distance between all plants that share an herbivore Symons 2000). Early studies of diet breadth failed to species (i.e., the extent of overall clustering), while the consider the phylogenetic nonequivalence of taxonomic NTI measures the average minimal distance between ranks (e.g. families and orders), and the phylogenetic plants that share an herbivore species (i.e., the extent of diversity index and the clade dispersion index, in terminal clustering). particular, were proposed to address this problem (Symons and Beccaloni 1999). However, these indices METHODS measured relatedness in terms of the branching order, Community ecology not branch lengths, of phylogenies. Branch lengths are Leaf-chewing insects were collected from 62 plant especially critical for studies of phylogenetic dispersion in species representing 41 genera and 18 families (Table 1). ecological communities with an uneven distribution of Sampling effort was equalized across all host plants to closely related and distantly related species (Cavender- provide quantitative estimates of herbivore relative Bares et al. 2004). Consider lowland tropical rain forest abundance. Parataxonomists and village collectors tree communities, for example, which are often domi- surveyed 1500 m2 of foliage over nearly 1600 field-days nated by a relatively small number of highly species-rich and .6 3 104 tree inspections. Live insects were genera and families (Novotny et al. 2002). In such cases, subjected to feeding trials with fresh foliage of the plant narrow host specificity of herbivores has been invoked to species from which they were collected in the field. These explain the maintenance of high insect species richness, procedures are detailed in Novotny et al. (2002). We but this conclusion was reached with little regard for host recorded 961 species and 62 193 individuals feeding on plant relatedness (Basset 1992). the 62 host plant species. Additionally, 40 000 insects The analysis presented here builds on an earlier study that failed to feed on the plant from which they were (Novotny et al. 2002), expanding a New Guinea host collected were discarded. Local parataxonomists as- plant assemblage from 51 to 62 species and applying new signed feeding specimens to morphospecies (Basset et al. indices of phylogenetic dispersion to herbivore associa- 2000), and taxonomic specialists later identified known tions. The island of New Guinea is the third largest taxa. Details on plant and insect identification are remaining area of tropical forest wilderness in the world reported in Miller et al. (2003). One-quarter of all species and includes ;5% of global plant and insect diversity were identified to named species, and 44% were while occupying only 0.5% of the land area (Miller identified to genus, but taxonomic knowledge varied 1993). Our study site near Madang, on the north coast from group to group. For example, 90% of the of Papua New Guinea, includes ;150 tree species/ha Lepidoptera species were assigned to a genus, and 72% that measure .5 cm dbh, and species richness is were associated with a known species, while only 39% of dominated by approximately a dozen genera. beetles were assigned to genus and 19% to species. The We quantified the relationship between the herbivore locality, collection date, and host plant species for 37 972 community similarity of host trees and the phylogenetic mounted specimens are also available in our database. distance between hosts. We defined similarity as the Digital photographs of many species are archived and ratio of the number of herbivore species sharing two available online.8 Sampling included 388 species and hosts to the total number of herbivore species feeding on the pair of hosts. Phylogenetic distance between host species was based on DNA sequence divergence 8 hhttp://www.entu.cas.cz/png/index.htmli S64 GEORGE D. WEIBLEN ET AL. Ecology Special Issue

TABLE 1. Plant species and gene sequences included in a phylogenetic study of host use in a tropical insect herbivore community.

Species Code Family Order Clade GenBank Amaracarpus nymanii Valeton AMA Rubiaceae Gentianales euasterids 1 AJ002176 Artocarpus camansi Blanco ART Moraceae Rosales eurosids 1 AY289288 Breynia cernua (Poir.) Muell. Arg BRE Phyllanthaceae Malphigiales eurosids 1 AY374311 Casearia erythrocarpa Sleum. CAS Flacourtiaceae Malphigiales eurosids 1 AF206746 Celtis philippensis Blanco CEL Ulmaceae Rosales eurosids 1 D86309 Codiaeum ludovicianum Airy Shaw COD Euphorbiaceae Malphigiales eurosids 1 AY374312 Dolicholobium oxylobum K. Schum. DOL Rubiaceae Gentianales euasterids 1 AJ318445 Dracaena angustifolia Roxb. DRA Agavaceae Asparagales monocots AF206729 Endospermum labios Schodde END Euphorbiaceae Malphigiales eurosids 1 AY374313 Eupomatia laurina R. Br. EUP Eupomatiaceae Magnoliales basals L12644 Excoecaria agallocha L. EXC Euphorbiaceae Malphigiales eurosids 1 AY374314 Ficus bernaysii King BER Moraceae Rosales eurosids 1 AF165378 Ficus botryocarpa Miq. BOT Moraceae Rosales eurosids 1 AF165379 Ficus conocephalifolia Ridley CON Moraceae Rosales eurosids 1 AF165381 Ficus copiosa Steud. COP Moraceae Rosales eurosids 1 AF165382 Ficus dammaropsis Diels DAM Moraceae Rosales eurosids 1 AF165383 Ficus hispidioides S. Moore HIS Moraceae Rosales eurosids 1 AF165388 Ficus microcarpa L. MIC Moraceae Rosales eurosids 1 AF165393 Ficus nodosa Teysm. & Binn. NOD Moraceae Rosales eurosids 1 AF165395 Ficus phaeosyce Laut. & K. Schum. PHA Moraceae Rosales eurosids 1 AF165401 Ficus pungens Reinw. ex Bl. PUN Moraceae Rosales eurosids 1 AF165404 Ficus septica Burm. f. SEP Moraceae Rosales eurosids 1 AF165409 Ficus tinctoria Forst. TIN Moraceae Rosales eurosids 1 AF165413 Ficus trachypison K. Schum. TRA Moraceae Rosales eurosids 1 AF165414 Ficus variegata Bl. VAR Moraceae Rosales eurosids 1 AF165415 Ficus wassa Roxb. WAS Moraceae Rosales eurosids 1 AF165418 Gardenia hansemannii K. Schum. GAR Rubiaceae Gentianales euasterids 1 AJ318446 Gnetum gnemon L. GNE Gnetaceae Gnetales outgroup AY056577 Homalanthus novoguineensis (Warb.) K. Schum. HON Euphorbiaceae Malphigiales eurosids 1 AY374315 Hydriastele microspadix (Becc.) Burret. ARE Arecaceae Arecales monocots AY012504 Kibara cf. coriacea (Bl.) Tul. STG Monimiaceae Laurales basals AF050221 Leucosyke capitellata (Poir.) Wedd. LEU Urticaceae Rosales eurosids 1 AY208707 Macaranga aleuritoides F. Muell. MAA Euphorbiaceae Malphigiales eurosids 1 AY374319 Macaranga bifoveata J. J. Smith MAP Euphorbiaceae Malphigiales eurosids 1 AY374321 Macaranga brachytricha A. Shaw MAF Euphorbiaceae Malphigiales eurosids 1 AY374316 Macaranga densiflora Warb. MAD Euphorbiaceae Malphigiales eurosids 1 AY374317 Macaranga novoguineensis J. J. Smith MAU Euphorbiaceae Malphigiales eurosids 1 AY374320 Macaranga quadriglandulosa Warb. MAQ Euphorbiaceae Malphigiales eurosids 1 AY374318 Mallotus mollissimus (Geisel.) Airy Shaw MAL Euphorbiaceae Malphigiales eurosids 1 AY374322 Melanolepis multiglandulosa (Reinw. ex Bl.) Reichb. f. MEL Euphorbiaceae Malphigiales eurosids 1 AY374323 Morinda bracteata Roxb. MOR Rubiaceae Gentianales euasterids 1 AJ318448 Mussaenda scratchleyi Wernh. MUS Rubiaceae Gentianales euasterids 1 AJ318447 Nauclea orientalis (L.) L. SAR Rubiaceae Gentianales euasterids 1 AJ318449 Neonauclea clemensii Merr. & Perry NEO Rubiaceae Gentianales euasterids 1 AJ318450 Neuburgia corynocarpa (A.Gray) Leenh. NEU Loganiaceae Gentianales euasterids 1 AJ001755 Osmoxylon sessiliflorum (Lauterb.) W.R.Philipson OSM Araliaceae Apiales euasterids 2 U50257 Pavetta platyclada Lauterb. & K. Schum. PAV Rubiaceae Gentianales euasterids 1 AJ318451 Phyllanthus lamprophyllus Muell. Arg. PHY Phyllanthaceae Malphigiales eurosids 1 AY374325 Pimelodendron amboinicum Hassk. PIM Euphorbiaceae Malphigiales eurosids 1 AY374324 Pometia pinnata Forster POM Sapindaceae Sapindales eurosids 2 AJ403008 Premna obtusifolia R.Br. PRE Verbenaceae Lamiales euasterids 1 U28883 Psychotria leptothyrsa Miquel PSF Rubiaceae Gentianales euasterids 1 AJ318452 Psychotria micralabastra (Laut. & Schum.) Val. PSM Rubiaceae Gentianales euasterids 1 AJ318453 Psychotria micrococca (Laut. & Schum.) Val. PSS Rubiaceae Gentianales euasterids 1 AJ318454 Psychotria ramuensis Sohmer PSL Rubiaceae Gentianales euasterids 1 AJ318455 Pterocarpus indicus Willd. PTE Fabaceae Fabales eurosids 1 AF308721 Randia schumanniana Merrill & Perry MEN Rubiaceae Gentianales euasterids 1 AJ318456 Sterculia schumanniana (Lauterb.) Mildbr. STR Malvaceae Malvales eurosids 2 AJ233140 Tabernaemontana aurantica Gaud. TAB Apocynaceae Gentianales euasterids 1 X91772 Tarenna buruensis (Miq.) Val. TAR Rubiaceae Gentianales euasterids 1 AJ318457 Timonius timon (Spreng.) Merr. TIT Rubiaceae Gentianales euasterids 1 AJ318458 Versteegia cauliflora (K. Schum. & Laut.) VER Rubiaceae Gentianales euasterids 1 AJ318459 Notes: When sequences were not available for particular species, substitutions of near relatives from GenBank were made (hhttp://www.ncbi.nlm.nih.govi). For example, rbcL sequences from Artocarpus altilis (AF500345) and Ficus heterophylla (AF500351) were substituted for ART and VAR, respectively. Additional substitutions are footnoted. Substituted rbcL sequences Amaracarpus sp. (AMA), Casearia sylvestris (CAS), Celtis sinensis (CEL), Agave ghiesbreghtii (DRA), Eupomatia bennetti (EUP), Gnetum parvifolium (GNE), Kibara rigidifolia (STG), Hydriastele wendlandiana (ARE), Urtica dioica (LEU), Teraplasandra hawaiensis (OSM), Talisia nervosa (POM), Premna microphylla (PRE), Willardia mexicana (PTE), Sterculia apetala (STR), and Tabernaemontana divaricata (TAB). July 2006 PHYLOGENY OF HOST ASSOCIATIONS S65

24 481 individuals for beetles (Coleoptera), 464 species for rbcL and 111 for ndhF yielded a rate of 1.914 for and 31 108 individuals for moths and butterflies (Lep- ndhF relative to rbcL. We rescaled the branch lengths by idoptera; see Plate 1), and 109 species and 6605 these rates to approximate the phylogenetic distance individuals of orthopteroids (Orthoptera and Phasma- between taxa sampled for genes showing radically todea). Among the caterpillars, ;14 000 were matched different rates of molecular divergence. The assumption with adults, amounting to 298 species of Lepidoptera of this method is that rates of divergence for each gene with known larval and adult stages. are homogeneous among the lineages comprising the community sample. In the case of plant families other Molecular phylogenetics than Moraceae, Rubiaceae, and Euphorbiaceae, rbcL Phylogenetic relationships for the 62 host plant sequences were not necessarily available from the species were drawn from multiple molecular data sets particular species, and in these instances sequences from including a three-gene phylogeny for all angiosperms related species or genera were obtained from GenBank (Soltis et al. 1998). We used additional molecular as indicated in Table 1. markers for species of Moraceae, Rubiaceae, and The next challenge is to obtain a phylogeny for Euphorbiaceae, including the internal transcribed spacer which all distances from the root of the tree to the tips (ITS) region of nuclear ribosomal DNA for Ficus are equal, also known as an ultrametric tree. Ultra- (Weiblen 2000), rbcL, encoding the large subunit of metricity is necessary to make direct comparisons of ribulose-1,5-bisphosphate carboxylase, and the 30S phylogenetic distance (as measured by rescaled molec- ribosomal protein S16 gene (rps16) for Rubiaceae ular branch lengths) among pairs of host species (Novotny et al. 2002), and ndhF, encoding a subunit distributed across the phylogeny. Each individual data of NADH-plastoquinone oxidoreductase, for the Eu- set rejected a molecular clock assumption, so we phorbiaceae. Phylogenetic analyses of Euphorbiaceae applied nonparametric rate smoothing and penalized ndh based on F are presented in the Appendix. likelihood as implemented in the program r8s (Sander- Community phylogenetics son 2002) to the rescaled branch lengths of the supertree to obtain an ultrametric tree accommodating A phylogeny estimate for the community sample was rate heterogeneity across lineages. Penalized likelihood obtained by grafting less inclusive single-gene phyloge- is a semiparametric method that allows substitution nies for Ficus, Euphorbiaceae, and Rubiaceae into a rates to vary among lineages according to a smoothing more inclusive phylogeny of angiosperms based on three parameter (Sanderson 2002). The optimal smoothing genes (Soltis et al. 1998). The assembly of a community parameter was chosen on the basis of the data by phylogeny can follow supertree methods (Sanderson et cross-validation involving the sequential pruning of al. 1998) or other approaches (Lapointe and Cucumel taxa from the tree and parameter estimation to best 1997), but one crucial difference is that only members of predict the branch length of the pruned taxon (Sander- the community are retained in the supertree, while all son 2003). We compared 20 cross-validation parame- other lineages are pruned away. It is important to consider the impact of branch length ters beginning with zero and increasing by increments of log10(0.05) and chose the optimal smoothing considerations on indices of phylogenetic clustering 2 drawn from community samples. When branch lengths parameter to minimize v error. Cross-validation was are assumed equal, using the number of intervening performed with the age of the root node fixed at one. nodes as a proxy for phylogenetic distance (Novotny et Penalized-likelihood search parameters included 2000 al. 2002), relationships between intensively sampled maximum iterations, 10 multiple starts, and 30 congeneric species are given the same weight as relation- optimization runs. ships among representatives of major clades. Branch Phylogenetic dispersion of herbivore associations length information can distinguish between these two very different cases, short distances between congenerics Herbivore associations with each of the 62 host and long distances between members of major lineages. species were coded as either present or absent under Therefore, to incorporate information from all three two different assumptions, including or excluding molecular data sets, we scaled branch lengths in the solitary observations. Where r denotes the number of supertree to the relative rate of change in two genes feeding records for a particular herbivore species on a compared between pairs of taxa. For example, the particular host species, associations were coded as relative rate of ITS to ndhF was calculated by counting present when r . 1 or when r . 0 to exclude or include the absolute number of character differences in each singletons, respectively. Varying this threshold allowed gene between Ficus microcarpa and F. variegata. us to examine the sensitivity of findings based on Including all characters, there were 15 ndhF differences presence/absence to extreme variation in herbivore between these species and 58 ITS differences, yielding a abundance. We examined the distribution of herbivore relative rate of 0.259 for ndhF to ITS (Weiblen 2000, associations across the host phylogeny, with indices of Datwyler and Weiblen 2004). Fifty-eight pairwise differ- phylogenetic clustering as implemented in the program ences between Artocarpus camansi and Ficus variegata Phylocom (Webb et al. 2004). S66 GEORGE D. WEIBLEN ET AL. Ecology Special Issue

FIG. 1. Phylogenetic relationships of host plant species included in the study (see Table 1 for abbreviations). Brackets indicate the three major angiosperm clades that were sampled intensively. A supertree was assembled from separate analyses of DNA sequences for Rubiaceae (Novotny et al. 2002), Ficus (Weiblen 2000), Euphorbiaceae (see the Appendix), and angiosperms as a whole (Soltis et al. 1998, Angiosperm Phylogeny Group 2003). Branch lengths based on ITS, rbcL, and ndhF sequences for partially overlapping sets of taxa were rescaled in proportion to pairwise differences between selected species with published ITS and ndhF, or ndhF and rbcL, sequences (see Methods). Branch lengths as shown are proportional to absolute numbers of nucleotide changes under parsimony. The scale bar indicates 10 changes.

The net relatedness index measured the mean distance for n host plants randomly distributed on the phylogenetic distance between all plants sharing a phylogeny, obtained by multiple iteration. The nearest particular herbivore: NRI ¼ –(Xnet – X(n))/SD(n) where taxon index measured the distance between the two Xnet is the mean phylogenetic distance between all pairs nearest hosts sharing a particular herbivore. This index of n host plants sharing an herbivore, and X(n) and SD(n) is calculated in the same manner as NRI, except that are the mean and standard deviation of phylogenetic Xnear is substituted for Xnet, where Xnear is the shortest July 2006 PHYLOGENY OF HOST ASSOCIATIONS S67

FIG. 2. Molecular divergence among 62 selected, woody host plant species in lowland tropical rain forest on the island of New Guinea (see Table 1 for species abbreviations). The ultrametric tree was derived from penalized-likelihood analysis. (a) Shallowest split between families, Loganiaceae and Apocynaceae, (b) deepest crown radiation of a genus, Psychotria, and (c) shallowest crown radiation of a genus, Ficus. Brackets mark the angiosperm families and genera that were the focus of herbivore sampling. Branch lengths as shown are proportional to the number of nucleotide changes per site under maximum likelihood. The scale bar indicates 0.05 substitutions per site. distance between all pairs of n host plants sharing an random association, we performed 1000 permutations herbivore. High values of these indices suggest cluster- of host associations to simulate a distribution of NRI ing, whereas low values point to evenness (i.e., over- and NTI for each herbivore species. A two-tailed test of dispersion). We tested whether these measures of significance evaluated the rank of observed values at P ¼ phylogenetic dispersion of herbivore associations across 0.05. For example, a rank of ,25 or .975 of 1000 the community phylogeny were significantly different permutations constituted significant overdispersion or from chance expectations. Under a null model of clustering, respectively. S68 GEORGE D. WEIBLEN ET AL. Ecology Special Issue

TABLE 2. Numbers and percentages of insect herbivore species with signiﬁcantly clustered (and overdispersed) patterns of host association across a community sample of 62 woody plant species from New Guinea lowland rain forest.

Excluding singletons NRI NTI Taxon NB BL PL LF NB BL PL LF Lepidoptera 76 (0) 65 (1) 61 (5) 61 (5) 68 (0) 58 (3) 60 (9) 60 (9) Lepidoptera (%) 55 (0) 47 (1) 45 (4) 45 (4) 50 (0) 42 (2) 44 (6) 44 (6) Coleoptera 30 (1) 43 (0) 46 (1) 46 (1) 26 (0) 27 (0) 24 (3) 24 (1) Coleoptera (%) 30 (1) 43 (0) 46 (1) 46 (1) 26 (0) 27 (0) 24 (3) 24 (1) Orthopteroids 12 (0) 14 (0) 9 (0) 9 (0) 8 (0) 4 (1) 4 (2) 4 (6) Orthopteroids (%) 30 (0) 35 (0) 22 (0) 22 (0) 20 (0) 10 (2) 10 (5) 10 (15) Total herbivores 118 (1) 122 (1) 116 (6) 116 (6) 102 (0) 89 (4) 88 (14) 88 (16) Herbivores (%) 43 (1) 44 (1) 42 (2) 42 (2) 37 (0) 32 (1) 32 (5) 32 (6) Notes: Two-tailed tests of phylogenetic dispersion assessed signiﬁcance at P ¼ 0.05 with ranks .975 (or ,25) out of 1000 randomizations. Abbreviations: NRI ¼ net relatedness index; NTI ¼ nearest taxon index; NB ¼ no. branch lengths (no. intervening nodes); BL ¼ rescaled molecular branch lengths (nonultrametric); PL ¼ rescaled ultrametric branch lengths (penalized likelihood); LF ¼ rescaled ultrametric branch lengths (Langely-Fitch nonparametric rate smoothing).

We further examined the relationship of herbivore rescaled in terms of ndhF substitutions. Nonparametric community similarity to the phylogenetic distance rate smoothing (Langely-Fitch) and penalized like- between hosts. We calculated community similarity as lihood yielded highly similar ultrametric trees (Fig. 2). the percentage of the total number of herbivore species As expected, phylogenetic distances between congeneric feeding on any pair of host species that were shared species were lower than between confamilial genera and between the hosts (Novotny et al. 2002). We estimated extaordinal families. phylogenetic distance from branch lengths based on DNA sequence divergence under penalized likelihood as Phylogenetic dispersion implemented in r8s (Sanderson 2002). We used linear Each of 226 Lepidoptera, 212 Coleoptera, and 87 regression to analyze the direction and linear regression orthopteroid species observed on multiple hosts was and Mantel tests to assess the significance of this tested for nonrandom patterns of association with relationship. respect to host plant phylogeny. Under a more stringent coding of host association that excluded all RESULTS solitary feeding records, the 137 Lepidoptera, 99 Community ecology Coleoptera, and 40 orthopteroid species encountered Among the 62 193 insects, including 464 caterpillar on multiple hosts (multiple times each) were also species reared to adults, 388 beetle species, and 109 analyzed with respect to host phylogenetic dispersion. orthopteroids, there were 281 species collected as single Results under four different branch length assumptions, individuals (singletons). Singleton species were excluded two different indices of phylogenetic dispersion, and from subsequent analyses, because it is impossible to two feeding thresholds indicated that herbivores with assess host range when a species is known from only one nonrandom dispersion of associations feed on closely feeding record (Novotny and Basset 2000). Apart from related hosts more often than on distantly related hosts singletons, our sample also included 156 herbivore (Table 2). In particular, 25–43% of the herbivore species that fed on a single plant species. Our analysis species we analyzed were significantly clustered on the did not examine whether these species are truly mono- host plant phylogeny compared to 0–6% that were phagous or were simply sampled in insufficient numbers. overdispersed. Rather, we focused on the host phylogenetic distribution The incorporation of sequence divergence in branch of associations for the remaining 524 herbivore species length estimation had a dramatic impact on the (55% of the total) that were found to feed on more than detection of phylogenetic dispersion. In the case of one plant species. nearest taxon index, for example, results under the assumption of equal branch lengths only agreed with Community phylogeny those under molecular branch length assumptions in A phylogeny was obtained for the host plant 65% of cases, three variations on the latter agreed in 93% community sample by grafting hypotheses of relation- of cases, and the two assumptions based on ultrametric ship for selected Euphorbiaceae (see Appendix). Rubia- trees agreed in all cases. Exclusion of feeding records ceae (Novotny et al. 2002), and Ficus (Weiblen 2000) to represented by single observations also enhanced the an ordinal phylogeny based on multiple data sets detection of nonrandom associations with respect to (Angiosperm Phylogeny Group 2003). The phylogeny host plant phylogeny. Without singletons, 32–37% of is shown in Fig. 1 with rbcL and ITS branch lengths herbivore species rejected the null model of association July 2006 PHYLOGENY OF HOST ASSOCIATIONS S69

TABLE 2. Extended.

Including singletons NRI NTI NB BL PL LF NB BL PL LF 124 (0) 93 (0) 90 (4) 91 (4) 103 (0) 78 (7) 75 (13) 76 (13) 55 (0) 41 (0) 40 (2) 40 (2) 46 (0) 34 (3) 33 (6) 34 (6) 41 (6) 70 (0) 75 (3) 76 (3) 45 (0) 44 (0) 49 (1) 50 (1) 19 (3) 33 (0) 35 (1) 36 (1) 21 (0) 21 (0) 23 (0) 23 (0) 20 (1) 27 (0) 23 (0) 24 (1) 13 (0) 8 (3) 9 (2) 10 (2) 23 (1) 31 (0) 26 (0) 27 (1) 15 (0) 9 (3) 10 (2) 11 (2) 185 (7) 190 (0) 188 (7) 191 (8) 161 (0) 130 (10) 133 (16) 136 (16) 35 (1) 36 (0) 36 (1) 36 (2) 31 (0) 25 (2) 25 (3) 26 (3)

compared to 25–31% including singletons in the extant taxa. This is why we applied indices of analysis, a trend that was upheld by each of three insect phylogenetic dispersion to examine the relationship groups. between herbivore associations and host plant phylogeny. Community similarity and phylogenetic distance Herbivore community similarity, defined as the frac- Phylogenetic dispersion of host associations tion of the total herbivore species on two host species that Community phylogenies, null models, and measures are shared between the hosts (Novotny et al. 2002), was of phylogenetic dispersion taken together increase the negatively associated with phylogenetic distance as precision with which herbivore associations can be estimated by rate-smoothed molecular divergence under studied. Previous attempts to quantify host specificity, penalized likelihood (Fig. 3). The regression of commun- for example, have either relied on taxonomic ranks that ity similarity against phylogenetic distance was highly are not commensurate with plant lineages or ignored the significant (ANOVA, F1, 3842 ¼ 1243.5, P , 0.0001), and branch length information contained in molecular the correlation between these variables was also signifi- phylogenies (Symons and Beccaloni 1999, Novotny et cant according to a Mantel test (Pearson’s product- al. 2002). Branch length assumptions influence our moment correlation, r ¼ 0.423, P , 0.01). Declining community similarity with increasing phylogenetic distance between hosts indicates that herbivores tend to feed on closely related plants more often than on distantly related plants.

DISCUSSION While it is tempting to trace ecological character evolution on community phylogenies, ancestral reconstructions of host associations in community samples often yield implausible inferences. Equally weighted parsimony for highly polyphagous species implies that these herbivores colonized the common ancestors of major angiosperm clades and were subsequently lost from some host lineages. Consider for example the ancestral association of Rhinoscapha tricolor under equally weighted parsimony (Fig. 4). It is highly unlikely that this particular polyphagous species was associated with the common ancestor of the angiosperms and the gymnosperm Gnetum. Ancestral state reconstructions are sensitive to taxon sampling (Cunningham et al. 1998, FIG. 3. Herbivore community similarity as a function of the Cunningham 1999), and colonization or extinction phylogenetic distance between host plants. Similarity is the patterns cannot necessarily be inferred from local fraction of the total fauna on two hosts that is shared between assemblages because community phylogenies are incom- the hosts. Phylogenetic distance was derived from the penal- plete by deﬁnition. This problem is not unique to the ized-likelihood ultrametric phylogram shown in Fig. 2. Means, standard deviations, and ranges of community similarity are evolution of host associations, but also occurs whenever shown for selected distance intervals. The outgroup is excluded the included taxa might be a subset of an entire clade of from the regression. S70 GEORGE D. WEIBLEN ET AL. Ecology Special Issue

nodes in the community phylogeny are assumed to be equidistant when they are not. Branch lengths scaled to molecular divergence distinguish between these cases and enhance the power to detect significant patterns in host use (Table 2). Ultrametric molecular branch lengths approximate relative ages of lineages, and thus the length of time for ecological associations or adaptations to arise. We found that a large proportion of herbivores feed on closely related plants, including congeneric species and confamilial genera, and that a small number of herbivores feed on more divergent hosts than expected by chance. The former pattern is expected in cases of herbivore specialization (Jaenike 1990, Futuyma et al. 1993) and the latter pattern when herbivores are tracking convergent chemical, morphological, or ecological host traits (Becerra 1997). The incorporation of molecular branch lengths in a community phylogeny assembled from multiple genes poses interesting methodological challenges that invite further exploration. Communities are usually composed of heterogeneous taxa, some very closely related and others distantly so. Grafting of multiple phylogenies based on different genes could be necessary when no single gene resolves phylogenetic relationships at all taxonomic levels in the community sample. This was the case in our sample, where ITS sequences were employed to resolve relationships among Ficus, but this region could not be aligned across plant families. Rescaling of branch lengths from different gene regions based on the ratio of absolute character differences between taxon pairs represents one possible solution among many. An improvement on our method would be to correct for multiple substitutions in a model-based maximum-likelihood framework when rescaling branch lengths across grafted phylogenies. We do not know the extent to which the phylogenetic dispersion of herbivores in our samples is representative of herbivore community structure on the complete local plant community or tropical rain forests in general. The scope of our sampling universe is incomplete for even the local community. Fifteen figs, 13 Rubiaceae, 13 Euphorbiaceae, and 21 other angiosperms hardly encompass the woody vegetation of a study area that contains hundreds of flowering plant species. The selection of study plants was made to replicate the taxonomic ranks of family and genus, and is at best a FIG. 4. Erroneous inference of ancestral host use in community samples under equally weighted parsimony. Rhino- highly skewed sample in terms of local abundance and scapha tricolor is a polyphagous generalist that parsimony distribution. At least one way to avoid artifacts due to suggests had an implausible, ancient association with the taxonomic unevenness is to restrict analyses to single common ancestor of Gnetum and flowering plants. See Table representatives of given taxonomic ranks, such as 1 for species abbreviations. families, but this is not satisfactory owing to the phylogenetic nonequivalence of taxa at any single rank. power to detect patterns of phylogenetic dispersion in at Age estimates of family clades in a recent study of least one important way. Failure to consider the extent angiosperms range from ,25 Ma to .150 Ma (Davies et of molecular divergence between hosts will under- al. 2004). The problem of taxonomic unevenness could estimate the extent of herbivore clustering (or over- be addressed by including all members of a local dispersion) given that closely related hosts and extremely community, provided that the boundaries of the divergent hosts with the same number of intervening community can be defined. We intend to explore these July 2006 PHYLOGENY OF HOST ASSOCIATIONS S71

FIG. 5. Phylogenetic dispersion of host range in 30 herbivore species arbitrarily selected from the community sample to illustrate the extremes of variation. Herbivores are grouped into nonsignificantly clustered species including polyphagous generalists, and significantly clustered species including oligophagous specialists feeding on Macaranga, Ficus,orPsychotria. Branch lengths of the host community phylogeny are proportional to molecular divergence as in Fig. 4, except for the truncated root indicated by a slash. Host species codes are defined in Table 1, and herbivore species codes are defined in Table 3. As in Fig. 4, solid boxes indicate herbivore presence and open boxes indicate herbivore absence. issues in the future through the complete enumeration of Novotny et al. (2002) to 62 in the present study, and vegetation in specific areas of forest (Novotny et al. through the incorporation of branch length information. 2004a). At the very least, it is encouraging that the It is remarkable that a full quarter of the variance of relationship between herbivore community similarity herbivore community similarity can be explained by the and host phylogenetic distance was strengthened by the phylogenetic relationships among hosts (r2 ¼ 0.244) expansion of our sample from 51 host species in when we consider the variability that environmental and S72 GEORGE D. WEIBLEN ET AL. Ecology Special Issue

TABLE 3. Herbivore species from Fig. 5 arranged alphabetically by morphospecies code.

Code Order Family Species NHNRI NTI ACRI001 Orthopteroid Pyrgomorphidae Desmopterella biroi (Bolivar, 1905) 2215 58 0.36 0.74 ACRI014 Orthopteroid Acrididae Valanga papuasica (Finot, 1907) 273 49 1.09 0.55 ACRI044 Orthopteroid Eumastacidae Paramnesicles buergersi Bolivar, 1930 111 29 1.28 1.94 ARCT002 Lepidoptera Arctiidae Darantasia caerulescens Druce, 1899 3 3 0.54 0.89 BUPR002 Coleoptera Buprestidae Habroloma sp. 20 3 3.34 2.33 CHOR008 Lepidoptera Choreutidae Brenthia salaconia Meyrick, 1910 389 7 5.60 2.38 CHRY004 Coleoptera Chrysomelidae genus indeterminate 125 6 6.60 2.60 CHRY076 Coleoptera Chrysomelidae Deretrichia sp. 16 5 5.01 2.45 CHRY124 Coleoptera Chrysomelidae Deretrichia sp. 16 6 0.55 0.26 CRAM003 Lepidoptera Crambidae Glyphodes margaritaria (Clerck) 1794 318 11 9.04 3.22 CRAM005 Lepidoptera Crambidae Talanga deliciosa (Butler) 1887 856 14 10.02 3.37 CRAM006 Lepidoptera Crambidae Talanga sexpunctalis (Moore) 1877 329 13 9.50 3.21 CRAM044 Lepidoptera Crambidae ‘‘Coelorhycidia’’ nr. purpurea Hampson, 1907 234 5 2.65 1.51 CURC002 Coleoptera Curculionidae Apirocalus ebrius Faust, 1892 2349 54 1.31 1.38 CURC005 Coleoptera Curculionidae Alcidodes elegans (Guerin) 1838 141 22 1.94 2.51 GEOM021 Lepidoptera Geometridae Cleora repetita Butler, 1882 12 9 0.11 0.21 LYCA006 Lepidoptera Lycaenidae Philiris helena Snellen, 1887 121 8 5.54 2.00 NOCT002 Lepidoptera Noctuidae Asota heliconia Linnaeus, 1758 257 9 7.70 2.94 NYMP001 Lepidoptera Nymphalidae Euploea leucosticos Gmelin, 1788 108 11 8.12 3.04 NYMP002 Lepidoptera Nymphalidae Cyrestis acilia Godart, 1819 156 12 9.52 3.32 PHAS002 Orthopteroid Heteronemiidae Neopromachus vepres (Brunner von Wattenwyl) 1907 211 41 0.15 1.43 PHAS004 Orthopteroid Phasmatidae Dimorphodes prostasis Redtenbacher, 1908 98 35 0.06 0.74 PHAS016 Orthopteroid Phasmatidae Eurycantha insularis Lucas, 1869 38 25 1.65 0.68 PSYC001 Lepidoptera Psychidae Eumeta variegata Snellen, 1879 33 19 0.72 0.12 PYRA002 Lepidoptera Pyralidae Orthaga melanoperalis Hampson, 1906 246 7 5.99 2.55 SPHI004 Lepidoptera Sphingidae Macroglossum melas Rothschild & Jordan, 1930 167 5 4.32 2.11 TORT006 Lepidoptera Choreutidae Choreutis lutescens (Felder and Rogenhofer) 1875 332 13 9.50 3.21 TORT008 Lepidoptera Tortricidae Adoxophyes templana complex 482 29 1.54 1.65 TORT040 Lepidoptera Tortricidae Homona mermerodes Meyrick, 1910 815 25 0.1 1.53 TORT075 Lepidoptera Thyrididae Mellea ramifera Warren, 1897 56 7 5.99 2.55 XXXX021 Lepidoptera Pyralidae Unadophanes trissomita Turner, 1913 301 5 4.97 2.35 XXXX048 Lepidoptera Gelechiidae Dichomeris ochreoviridella (Pagenstecher) 1900 394 6 5.79 2.48 XXXX076 Lepidoptera Gelechiidae Dichomeris sp. nr. resignata Meyrick, 1929 324 10 6.01 1.88 Notes: The total number of individuals (N) and the total number of host species (H), including solitary feeding records, are indicated. Net relatedness (NRI) and nearest taxon (NTI) indices are reported as calculated under the penalized-likelihood tree (Fig. 2). population demographical heterogeneity must inevitably Tests of host specificity contribute to samples of herbivore associations from Community phylogenies and null models provide a any site over any period of time. A recent community quantitative test of significance for host specificity at the phylogenetic analysis of host use by beetles in Panama- clade level. Examples of specialists on Macaranga, Ficus, nian rain forest revealed the same pattern (Ødegaard et and Psychotria that rejected null models of host al. 2005). These findings provide quantitative support association are shown in Fig. 5 and Table 3, along with for long-standing theory (Ehrlich and Raven 1964, nonspecialists that failed to reject null models. These Strong et al. 1984, Schoonhoven et al. 1998). There are examples were chosen to illustrate extreme cases and to at least two explanations for the decline in herbivore reinforce the point that a quantitative definition of host similarity with increasing phylogenetic distance between specificity based on phylogenetic dispersion is more hosts. The first has to do with the phylogenetic practical and powerful than definitions based on conservatism of host choice as manifest in the tendency arbitrary taxonomic ranks. When singleton records were for herbivore offspring to feed on the same host lineages excluded from analyses, more host clade specialists were detected in all herbivore assemblages (Table 2). This as their parents. Second, it is possible that host choice is result is not surprising given that herbivores tend to have influenced by the conservatism of chemical, morpho- a highly skewed distribution of abundance across the logical, ecological, and physiological plant traits affect- host range. The average herbivore species in New ing herbivore performance. Power to detect phylogenetic Guinea secondary forest, for example, has .90% of conservatism in community samples could be improved individuals aggregated on a single host species and feeds by considering species abundance and increasing the on one to three host species (Novotny et al. 2004b). universe of sampled hosts. Nonetheless, species pres- Singletons representing rare or anomalous feeding ence/absence and a limited sample of the local plant events are likely to increase error rates in analyses such community indicated a relatively strong influence of host as ours that ignore abundance distributions and simply relatedness on herbivore community composition. treat the associations as present or absent. July 2006 PHYLOGENY OF HOST ASSOCIATIONS S73

PLATE 1. Darantasia caerulescens Druce (Lepidoptera: Arctiidae): (A) Adult, (B) larva, and (C) male genitalia, with aedoeagus separated and vesica inﬂated. Genitalia, illustrated here for the ﬁrst time, allow differentiation from similar-looking species. The caterpillars of this moth fed on three distantly related plant species (Fig. 5). Photo and dissection credit: Karolyn Darrow.

Excluding singletons and considering molecular Clustering of similar plant traits in close relatives due branch lengths, the NRI and NTI differed as expected, to phylogenetic conservatism (Cavender-Bares et al. considering that NTI quantifies dispersion near the tips 2004) provides a simple explanation for the extreme of the phylogeny whereas NRI measures overall patterns of clade specialization observed in many dispersion. According to NRI, Lepidoptera was propor- herbivore species. We believe that herbivore tracking tionally the most specialized fauna, with 42–44% of of phylogenetically conserved plant traits is a more species significantly clustered with respect to host plant plausible explanation than co-cladogensis for patterns of phylogeny, compared with 24–27% of coleopterans and association in many plant-herbivore interactions. Pre- 10% of orthopteroids. By contrast grasshoppers and dation and parasitism might also indirectly promote relatives showed the highest proportion of overdispersed specialization in phytophagous insect communities species (2–15%), compared with Lepidoptera (2–6%) (Bernays and Graham 1988). Attack rates of caterpillar and Coleoptera (0–3%). We attribute these differences to parasitoids in temperate forests, for example, vary variation among feeding guilds. We expected Lepidop- among host plant species, and this variation has the potential to influence the evolution of herbivore host tera to show the greatest overall degree of host range (Lill et al. 2002). Apart from patterns of clade specificity, due to the fact that caterpillars feeding on specialization, we also detected a small number of cases foliage were reared from larvae to adults and host of phylogenetic overdispersion (3–6% of all herbivores) specificity is manifest at the larval stage. Coleoptera, on that could have a biological explanation. the other hand, were tested for feeding only as adults Significant overdispersion is expected for herbivores and potentially feed on a more restricted set of hosts as feeding on distantly related hosts when hosts share a set larvae. Root-, stem-, and wood-boring beetle larvae are of convergent traits that are palatable to particular expected to exhibit greater host specialization than adult herbivores (Cavender-Bares and Wilczek 2003). Con- stages, because the impact of plant chemistry on insect vergence in plant traits can result from adaptive evolution development is strongest in the early life stages (Mattson (Agrawal and Fishbein 2006) or habitat specialization et al. 1988). The nonholometabolous assemblage of (Fine et al. 2006). For example, Ficus tinctoria (Mor- grasshoppers and relatives, feeding as nymphs and aceae) and Excoecaria agallocha (Euphorbiaceae) share adults, are widely regarded as polyphagous (Chapman an extreme environment and a unique set of herbivores and Sword 1997) and therefore expected to show less along the seacoast. The identification of convergent phylogenetic clustering and greater overdispersion than ecophysiological, morphological, and chemical traits in the other assemblages. Orthopteroid nymphs are more distantly related hosts sharing similar herbivores might mobile than caterpillars, enhancing opportunities to point to factors that limit the evolution of host range. graze on multiple hosts and presumably selection for Where trait convergence enables similar insects to feed on greater breadth of diet (Chapman and Sword 1997). highly diverged plant lineages, we expect significant S74 GEORGE D. WEIBLEN ET AL. Ecology Special Issue herbivore clustering in more than one place on the plant Becerra, J. X. 1997. Insects on plants: macroevolutionary phylogeny, causing the nearest taxon index to be chemical trends in host use. Science 276:253–256. Bernays, E., and M. Graham. 1988. On the evolution of significantly high when the net relatedness index is not. phytophagous arthropods. Ecology 69:886–892. Cavender-Bares, J., D. D. Ackerly, D. A. Baum, and F. A. Conclusions Bazzaz. 2004. Phylogenetic overdispersion in Floridian oak This study of herbivore associations illustrates how communities. American Naturalist 163:823–843. the integration of community ecology and phylogeny Cavender-Bares, J., and A. Wilczek. 2003. Integrating micro- and macroevolutionary processes in community ecology. can detect patterns of host specialization. A community Ecology 84:592–597. phylogeny with molecular branch lengths and null Chapman, R. F., and G. A. Sword. 1997. 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APPENDIX A description of Euphorbiaceae phylogeny (Ecological Archives E087-111-A1). Ecology, 87(7) Supplement, 2006, pp. S76–S85 Ó 2006 by the Ecological Society of America

ECOLOGICAL FITTING AS A DETERMINANT OF THE COMMUNITY STRUCTURE OF PLATYHELMINTH PARASITES OF ANURANS

1,5 2 3 4 DANIEL R. BROOKS, VIRGINIA LEOŃ-RE` GAGNON, DEBORAH A. MCLENNAN, AND DEREK ZELMER 1Department of Zoology, University of Toronto, Ontario M5S 3G5 Canada 2Laboratorio de Helmintologıá, Instituto de Biologıá, Universidad Nacional Autońoma de Me´xico, C. P. 04510, D. F. Me´xico, Me´xico 3Department of Zoology, University of Toronto, Ontario M5S 3G5 Canada 4Department of Biological Sciences, Emporia State University, Emporia, Kansas 66801 USA

Abstract. Host–parasite associations are assumed to be ecologically specialized, tightly coevolved systems driven by mutual modification in which host switching is a rare phenomenon. Ecological fitting, however, increases the probability of host switching, creating incongruences between host and parasite phylogenies, when (1) specialization on a particular host resource is a shared characteristic of distantly related parasites, and (2) the resource being tracked by the parasite is widespread among many host species. We investigated the effect of ecological fitting on structuring the platyhelminth communities of anurans from a temperate forest and grassland in the United States and tropical dry and wet forests in Mexico and Costa Rica. The six communities all exhibit similar structure in terms of the genera and families inhabiting the frogs. Parasite species richness is highly correlated with the amount of time a host spends in association with aquatic habitats, a conservative aspect of both parasite and host natural history, and determined in a proximal sense by host mobility and diet breadth. The pattern of parasite genera and families within host genera across the regions examined is consistent with the prediction that ecological fitting by phylogenetically conservative species, coupled with historical accidents of speciation and dispersal, should be evidenced as a nested- subset structure; the shared requirement for aquatic habitats of tadpoles provides a baseline assemblage to which other parasite taxa are added as a function of adult host association with aquatic habitats. We conclude that parasite communities are structured by both ecological fitting and coevolution (mutual modification), the relative influences of which are expected to vary among different communities and associations. Key words: anurans; coevolution; community structure; Costa Rica; ecological fitting; frogs; Mexico; nested subset; parasitic platyhelminths; phylogenetic conservatism; toads; United States of America.

INTRODUCTION cesses, nor is there an expectation of complete con- There are two approaches to studying the evolution of gruence. Brooks proposed that the incongruent portions host–parasite associations. The first and newer research of host–parasite phylogenies falsified the hypothesis of program, maximum co-speciation, assumes that hosts co-speciation at those nodes and thus required inves- and their parasites share such a specialized and exclusive tigations into the influence of other factors (e.g., dispersal and host switching) on the evolution of the evolutionary association (Page 2003, Clayton et al. 2004, association. For example, parasites might diverge more Johnson and Clayton 2004) that speciation in one rapidly than their hosts via sympatric speciation, pro- lineage causes speciation in the other (synchronous co- ducing sister species inhabiting the same host (Brooks speciation; Hafner and Nadler 1988, 1990). Host– and McLennan 1993; or ‘‘lineage duplication’’ sensu parasite phylogenies are thus expected to be completely Page [2003]), or ecological or immunological evolution congruent, with departures from congruence explained in the host lineage could cause parasite extinction by invoking extinction in one lineage or the other. The (lineage sorting or ‘‘missing the boat’’ sensu Page second and original research program (Brooks 1979) is [2003]). also based upon comparing host–parasite phylogenies Although the maximum co-speciation program has and identifying points of congruence as instances of co- been moving closer to Brooks’ propositions about the speciation (the term coined by Brooks in [1979]). There way incongruences should be treated, there is still one are, however, no assumptions about underlying pro- area of dispute between the two perspectives, the importance of host switching during the evolution of Manuscript received 20 January 2005; revised 12 September host–parasite associations. This debate is a logical 2005; accepted 21 September 2005. Corresponding Editor (ad extension of the assumption that hosts and parasites hoc): C. O. Webb. For reprints of this Special Issue, see footnote 1, p. S1. share a specialized exclusive evolutionary association, 5 E-mail: [email protected] making it extremely unlikely that a parasite could S76 July 2006 ECOLOGICAL FITTING OF ANURAN PARASITES S77

PLATE 1. Major habitat types in Area de Conservacion Guanacaste, Costa Rica (clockwise from upper left); Pitilla approach, tropical cloud forest habitat at entrance to Pitilla Field Station; Cuajiniquil approach, Santa Elena Peninsula, seen from road to Cuajiniquil, tropical dry forest habitat; Rio Pizote, permanent swamp near Rio Pizote, between Dos Rios and Brasilia, tropical rain forest habitat. Pitilla Forest, tropical cloud forest habitat near Pitilla Field Station. All photos were taken in June 2005. Photo credits: D. R. Brooks change host species. This assumption, however, arises functions (or both), then the stage is set for the from believing that it is the host species, not a biological appearance of ecological specialization and close (co)- characteristic or combination of characteristics of the evolutionary tracking as well as host switching. Eco- host, that is important to the parasite (Brooks and logical fitting thus explains how a parasite can be McLennan 1993). Once researchers began thinking in ecologically specialized and still switch hosts: if the terms of traits rather than taxonomy, it became evident resource is widespread across many host species, then that parasites might be able to switch hosts if the trait the parasite can take advantage of an opportunity to they were tracking was shared among two or more hosts. establish a ‘‘new’’ specialized association without the cost The fact that present-day associations might be shaped of evolving novel abilities (Brooks and McLennan 2002). in part by the distribution of phylogenetically conserva- Just because a resource is widespread does mean that tive traits is called ecological fitting (Janzen 1985). it is automatically available. The geographic distribution There are many macroevolutionary manifestations of of the parasite might not coincide with the geographic ecological fitting. For example, any given parasite distribution of all hosts having the resource (Pellmyr species might be a resource specialist, but also might 1992a, b), or some other aspect of host biology might share that specialist trait with one or more close make the resource inaccessible to the parasite. For relatives. That is, specialization on a particular resource example, if host species A bearing resource x is highly can be plesiomorphic within a group (for an extensive abundant in a community, then less-abundant host discussion and examples see Brooks and McLennan species B and C, which also bear x might not be [2002]). On the other hand, the resource itself might be ‘‘apparent’’ to a parasite specializing on that resource at once very specific and taxonomically and geograph- (Feeny 1976, Wiklund 1984, Courtney 1985). Such ically widespread if it is a persistent plesiomorphic trait density-dependent factors provide the appearance of in the hosts. The evolutionary basis for ecological fitting close ecological tracking between the parasite and is thus deceptively simple, yet powerful. If specific cues/ species A at time T0. If some environmental stressor resources are widespread, or if traits can have multiple later decreases the abundance of species A, and C S78 DANIEL R. BROOKS ET AL. Ecology Special Issue

FIG. 1. Ecological fitting in frog lung flukes. A clade of lung flukes (Haematoloechus spp.) arose in conjunction with the evolution of leopard frogs (rpg; Rana pipiens group). Haematoloechus floedae arose through speciation by host switching to bullfrogs (bg; Rana catesbeiana). Haematoloechus floedae was introduced into Costa Rica with bullfrogs, where it expanded its host range to include local species of leopard frogs, members of the ancestral host group. Bullfrogs subsequently died out in Costa Rica, but H. floedae survives today due to ecological fitting (from Brooks et al., in press). Thick lines indicate episodes of cospeciation; thin lines indicate episodes of speciation by host switching. becomes relatively more apparent, then the parasite will Yucatań, and Costa Rica, the parasite went with them, become associated with C at time T1. This manifestation and is now found in bullfrogs in those areas, as well as in of ecological fitting could explain seemingly rapid and leopard frogs in the Yucatań and Costa Rica. Leopard virtually unconstrained evolution of novel specialized frogs (Rana pipiens clade) are the plesiomorphic hosts host associations. Finally, a parasite might have a for Haematoloechus (Fig. 1). Although the ancestor of hierarchy of host preferences, even though it is tracking H. floedae switched to bullfrogs, the presence of the the same resource (host rank order; Singer et al. 1971; fluke in leopard frogs indicates that the parasite has Janz and Nylin 1998 and references therein). The retained its clade’s plesiomorphic ability to infect hierarchy arises because the costs of accessing the leopard frogs (Brooks et al., in press). Interestingly, resource might not be identical across all host species bullfrogs have disappeared from Costa Rica, but the or even across individuals in the same species (Singer et parasite persists, having survived the ‘‘extinction’’ of its al. 1992). Such costs will depend on many different preferred host. This is the first demonstration that factors, including concentration of the resource, host parasites, like phytophagous insects (Janz et al. 2001 and density, and difficulty in extracting the resource. Overall, references therein) might display ancestral host prefer- parasites accessing a plesiomorphically (or, less often, ences under certain circumstances. homoplasiously) distributed resource are ‘‘faux general- Ecological fitting is generally investigated in insect– ists’’ (Brooks and McLennan 2002): specialists whose plant systems, because researchers can reconstruct host range appears large, but who are in reality using the phylogenetic patterns of association between the two same resource. clades, then examine the processes underlying those If a parasite species evolves the ability to utilize a patterns by (1) identifying the resource being tracked by novel resource, a second and more complicated type of the insect, (2) determining the distribution of that host preference hierarchy can arise if the parasite also resource among host plants, and (3) delineating the retains sufficient information to use the plesiomorphic host preference hierarchy of the insects (Brooks and resource (Wiklund 1981, Courtney et al. 1989). For McLennan 2002). Currently, we do not have this degree example, Haematoloechus floedae is a fluke native to the of detailed information for any host–parasite system. It southeastern United States where it lives in the lungs of is possible, however, to take advantage of ‘‘natural the bullfrog, Rana catesbeiana. When bullfrogs were experiments’’ (e.g., the case of H. floedae), or even to introduced to the southwestern United States, the make inferences based on contemporary patterns of July 2006 ECOLOGICAL FITTING OF ANURAN PARASITES S79 host–parasite association, if hosts vary in their use of a of the 75 sampled anurans, only six of which (Rana habitat to which parasite species are constrained. The catesbeiana, Rana vaillanti, Smilisca baudenii, Smilisca associations between anurans and their platyhelminth phaeota, Leptodactylus melanonotus, and Bufo marinus) parasites provide a model system for such an inves- have been sampled in two areas, and one of which tigation, because the majority of helminths require water (Bufo marinus) was sampled in three areas. From this for the development and transmission of infective stages, we conclude that comparisons of compound commun- while most, but not all, major groups of anurans have a ity structure among the six sites will not be confounded sexual and developmental tie to aquatic habitats. Brandt by multiple samples of the same anuran community, (1936) suggested that species richness in anuran parasite and therefore the same anuran parasite (i.e., pseudo- communities was directly related to the amount of time replication). Moreover, given the geographical and the host spent in or near water, an observation taxonomic breadth of the surveys, it is assumed that confirmed by subsequent studies (Prokopic and Kriva- the resultant presence/absence matrices of host–parasite nec 1975, Brooks 1976). A shared plesiomorphic associations, at the taxonomic levels examined (i.e., requirement for an aquatic habitat, coupled with a host genera and parasite genera and families) are gradient of adult anuran preferences ranging from representative of the possible associations, and not aquatic to arboreal, suggests that ecological fitting as a strongly biased by ecological factors, such as host and determinant of the parasites associated with a given parasite ranges and relative abundances. anuran taxon should be evidenced as a nested-subset Anuran species were ranked based on their associa- structure (Patterson and Atmar 1986) of host–parasite tion with aquatic habitats as follows: 7, riparian, associations across anuran taxa (Zelmer et al. 2004). prolonged breeding (several months); 6, semiaquatic, At one extreme, if all the host–parasite associations prolonged breeding; 5, terrestrial, prolonged breeding; 4, are the result of ecological fitting, then all host taxa are terrestrial, explosive breeding (1–2 wk); 3, arboreal, interchangeable from the point of suitability for the prolonged breeding; 2, arboreal, explosive breeding; 1, parasites, and associations will be determined solely by fossorial. The relationship between the ranked associa- the habitats the host utilizes and its feeding preferences. tion and trematode species richness was evaluated using The shared requirement of tadpoles for aquatic habitats Spearman’s rank correlation analysis. should thus provide a baseline assemblage of parasites Without data from experimental infections, ecological that infect the tadpole stage, while the parasites of adult fitting and co-speciation cannot be distinguished as anurans should accumulate in anuran host species as a explanations for extant, and apparently specific, host– function of the time they spend in aquatic habitats as parasite associations. Thus, parasite species and host adults. If specialized coevolutionary processes dominate, species were grouped by genera for the purpose of sympatry between anurans and the infective parasitic nested-subset analysis, increasing the likelihood that the stages will result in parasitism of only appropriate hosts, host and parasite clades had at one time been sympatric. producing idiosyncratic (i.e., ‘‘unexpected’’) presences Given the degree of local adaptation for both the host and absences in the matrix of host–parasite associations. and parasite species, pooling hosts by genera and parasites by genera and families should not increase MATERIALS AND METHODS the likelihood of a nested-subset pattern occurring, given Compound parasite communities are defined as the a mechanism of co-speciation. Thus it is necessary to array of parasite species inhabiting an array of host view such a pattern as having been produced by species in a given area (Holmes and Price 1986). We ecological fitting. Examination of the nested-subset have data for six compound communities of platyhel- structure of parasite genera within the pooled anuran minths that parasitize frogs as definitive hosts in North genera across all six localities was conducted using the and Central America: the temperate hardwood forests of nestedness temperature calculator (Atmar and Patterson North Carolina (Brandt 1936), the temperate grasslands 1995), which calculates the temperature of the matrix (a of Nebraska (Brooks 1976), and the tropical wet and dry measure of order, with lower temperatures indicating a forests of Costa Rica (see Plate 1) and Mexico, derived higher degree of order) and idiosyncratic host and from biodiversity inventories currently being coordi- parasite temperatures, which indicate host species and nated by D. R. Brooks (Costa Rica) and V. Leoń- parasite species contributing disproportionately to the Re` gagnon (Mexico) (see the Appendix). lack of order in the matrix (Atmar and Patterson 1993). We sampled 75 anuran species in the six areas; 59 Nested-subset patterns can arise as artifacts of were sampled in one area, 14 species were sampled in random draws of individual items from categories that two areas, and two species were sampled in three areas vary in their representation (Connor and McCoy 1979). (see the Appendix, Table A1). Of the 57 platyhelminth In a proximal sense, within a given locality, this would species collected, 38 were found in one area, 13 species involve host individuals acquiring parasites from a were found in two areas, four species were found in species pool where the probabilities of infection varied three areas, and two species (Langeronia macrocirra and among the parasite species because of an uneven Haematoloechus complexus) were found in four areas distribution of infective stages within the environment. (see the Appendix, Table A2). The parasites inhabit 34 Considering the patterns of association between host S80 DANIEL R. BROOKS ET AL. Ecology Special Issue and parasite taxa, assuming that the various host taxa species. For the RELABUND model considering para- are sympatric in a regional sense, nestedness could be site families, eight ‘‘individual’’ lecithodendriids are expected to arise by a similar passive mechanism if the distributed randomly among the host taxa.) As with parasite taxa vary in the degree of sympatry between the interpretation of these models for nestedness in their respective geographic ranges and those of the communities, some of the associations observed will be hosts. For tests of passive sampling involving commun- the result of host capture, and some by inheritance, ity data, the relative abundances of the species sampled placing the appropriate, but unknown, null model is not known for the source pool, requiring estimation of between the extremes. Both the RANDOM1 and these relative abundances from the available data. RELABUND null models were applied to presence/ Models that test for passive sampling typically base this absence matrices of platyhelminth genera within anuran estimate on the occurrence of species in the sample genera, trematode genera within anuran genera, and (RANDOM1; Patterson and Atmar 1986, Fischer and matrices where parasite genera not represented in all Lindenmayer 2002), which will result in overestimation three regions were pooled by family and evaluated of the colonization probabilities of rare species unless within host genera and host ecotype ranking. none of the populations present in the sample were To evaluate whether the patterns of presence and further supplemented by dispersal from the source pool absence revealed across regions by the nested-subset following the initial colonization (Andreń 1994). analysis were reflected at smaller scales, we employed Constructing an appropriate null model for passive Spearman’s rank correlation analysis to assess co- sampling would require knowledge of the contribution variance between the total number of host genera of immigration from the source pool to the observed occupied by a parasite genus or family (pooled across relative abundances. In the absence of such information, all six localities), and the number of host species, genera, a null model (RELABUND) defining the opposite and families occupied by each species within that taxon extreme, i.e., each individual present in a population is within each of the six localities. We also used Pearson’s assumed to be an immigrant from the source pool, can analysis to determine covariance between the total be used in concert with RANDOM1, with the appro- number of host genera occupied by a parasite genus or priate, but unavailable, null model falling between these family (pooled across all six localities) and the number extremes (Zelmer et al. 2004). Given that the distribu- of host species, genera, and families occupied by those tions of temperatures of matrices produced by these taxa within each region (United States, Mexico, and models represent extremes in terms of the effect of Costa Rica). immigration on the observed population sizes within a community, overlap with the tails of these distributions RESULTS cannot be evaluated with a simple decision rule and The ranked association with aquatic habitats of the must be interpreted in light of ecological evidence for the anuran species with nine or more individuals necropsied expected effects of immigration. per locality positively covaried (r ¼ 0.785; P , 0.0001) By analogy, the evaluation of passive mechanisms with the trematode richness of the frog host species that produce nested-subset patterns of associations (Fig. 2), with no clear differences in the pattern of between host taxa and parasite taxa would require an increase among the six localities. understanding of the contribution of host capture to the The temperature (the measure of matrix order derived observed associations. Species-level host and parasite by Atmar and Patterson [1993]) of the presence/absence phylogenies do not yet exist for the taxa in question (an matrix of platyhelminth genera within anuran genera exception is Haematoloechus;Leoń-Re` gagnon and (Fig. 3) was significantly more ordered than the matrices Brooks 2003), so the number of times a particular host produced by the RANDOM1 (P ¼ 0.00063) or genus acquired any particular parasite genus or family RELABUND (P ¼ 0.00002) null models. Nested-subset cannot be directly inferred, and must be estimated from analysis designated four of the 21 parasite taxa as the available presence/absence data. Analogous models idiosyncratic; two monogenean genera and two cestode to RANDOM1 and RELABUND were employed, using genera. Such idiosyncrasies are an indication of different the occurrence of parasite taxa within host taxa to colonization histories for these genera (Atmar and parameterize the Monte Carlo simulations for RAN- Patterson 1993) relative to the other parasites consid- DOM1, and using the number of independent host– ered, suggesting the importance of phylogenetic con- parasite associations to parameterize RELABUND. gruence as a determinant of the anuran associations with (For example, there are two species of Langeronia, one monogenean and cestode species. Consequently, the infecting four host species, the other infecting one. Thus, remaining analyses focused on the trematodes. for the RELABUND model considering parasite genera, The nested-subset structure of the trematode genera five ‘‘individuals’’ of Langeronia are distributed ran- within the pooled anuran genera (Fig. 4) also was domly among the host taxa. Within the Lecithodendriid significantly colder than the matrices generated from family, in addition to the associations mentioned for both null models (RANDOM1, P ¼ 0.000001; RELA- Langeronia, there are two other parasite species, one BUND, P ¼ 0.0000004), and also revealed idiosyncratic infecting two host species, and one infecting a single parasite genera. These idiosyncrasies all occurred in July 2006 ECOLOGICAL FITTING OF ANURAN PARASITES S81

FIG. 2. Increasing trematode species richness in anuran host species from each of six localities, with increased anuran association (ranked) with aquatic habitats. Multiple observations at a single coordinate are indicated parenthetically above the coordinate. ACG denotes Area de Conservacioń Guanacaste. genera that were missing from at least one of the three the caveat that the distribution of the appropriate null regions sampled (North America, Mexico, or Costa model presumably has a warmer central tendency than Rica). Thus, in order to evaluate the potential for the that produced by the RELABUND model. interaction of the anuran host genera with specific The total number of host genera occupied by a landscapes to produce nested subset patterns, we parasite genus or family (pooled across all six localities) arbitrarily pooled parasite genera not represented in all positively covaried with the number of host species (r ¼ three areas into their respective families, thereby 0.321; P ¼ 0.0104), genera (r ¼ 0.425; P ¼ 0.0005), and ensuring that all anuran genera included in the analysis families (r ¼ 0.426; P ¼ 0.0005) occupied by each species have the potential to draw from the same parasite pool. within that taxon within each of the six localities. The The resulting matrix is depicted in Fig. 5. total number of host genera occupied by a parasite The temperature of the presence/absence matrices of genus or family also positively covaried with the number trematode genera/families in pooled anuran genera of host species (r ¼ 0.492; P ¼ 0.0147), genera (r ¼ 0.668; overlaps the cold tail of the distribution produced by P ¼ 0.0004), and families (r¼ 0.645; P ¼0.0007) occupied the RANDOM1 model (P ¼ 0.0025) and the cold tail of by each species within that taxon within each of the the distribution produced by the RELABUND model three regions (United States, Mexico, and Costa Rica). (P ¼ 0.0885). Thus, the observed nested-subset pattern could only be attributed to passive mechanisms by DISCUSSION adhering to the RELABUND model’s assumption that Parasite habitat preference and transmission patterns all host–parasite associations occur independently. Given that extreme assumption, however, interpreta- Fifty-four of the 57 parasite species (see the Appendix, tions of the observed matrix as nonsignificant with Table A3) exhibit the plesiomorphic pattern of requiring regards to passive sampling must be made with caution. water for transmission, either by utilizing aquatic Examination of the presence/absence matrix of trem- intermediate hosts (digeneans and cestodes), or by atode genera and families within ranked anuran ecotypes swimming from one host to another (monogeneans). (Fig. 6) supports the contention that the semiaquatic In other words, transmission patterns are phylogeneti- anuran habitat creates overlap with infective stages of a cally conservative in this phylum (Brooks and greater number of trematodes than a purely aquatic McLennan 1993, Adamson and Caira 1994). This habit. The temperature of this matrix falls within the cold explains why 45 of the 57 platyhelminth species were tail of the distribution of the temperatures produced by found only in aquatic and semiaquatic frogs. Of the both models (RANDOM1, P ¼ 0.0018; RELABUND, P remaining 12 species, 10 occur in terrestrial, arboreal, ¼ 0.165). As with the parasite associations with anuran and fossorial frogs, but infect the tadpole stage of their genera, one must conclude that passive mechanisms could hosts. Digeneans in the genus Glypthelmins and the produce this pattern only under the independent-associ- family Paramphistomidae cluster with the brachycoelids ation assumption of the RELABUND model, again with (Brachycoelium and Mesocoelium) in the maximally S82 DANIEL R. BROOKS ET AL. Ecology Special Issue

FIG. 3. Maximally packed presence/absence matrix for pooled parasite genera (columns) within pooled host genera (rows) from all six localities. Stars indicate idiosyncratic hosts and parasite species. Letters within the matrix denote geographic region where associations were observed: A, United States only; B, Mexico only; C, Costa Rica only; D, United States and Mexico; E, United States and Costa Rica; F, Mexico and Costa Rica; G, United States, Mexico, and Costa Rica. packed matrix (Fig. 5) as parasite taxa common to a Choledocystus intermedius (one of the idiosyncratic number of anuran genera within localities and regions, taxa in the presence/absence matrix depicted in Fig. 4) as well as across regions. All that is required for in- inhabits only Bufo marinus, and it is the only adult platy- fection is that the frog species comes to water to breed in helminth restricted to that host. Razo-Mendivil et al. (in a population density high enough to ensure infection. press) recently have shown C. intermedius to be closely This behavior is plesiomorphic for, and phylogenetically related to members of the families Ochetosomatidae and conservative among, frogs. The last two species, Telorchiidae. Life cycles for members of those families members of the sister groups Brachycoelium and involve aquatic molluscs as ﬁrst intermediate hosts, and Mesocoelium, have terrestrial life cycles, which explains tadpoles as second intermediate hosts, which are ingested why they occur so frequently in terrestrial anurans and by the ﬁnal host. The absence of C. intermedius from other in frogs that occasionally forage away from water. anuran hosts that ingest tadpoles might indicate that the

FIG. 4. Maximally packed presence/absence matrix for pooled trematode genera (columns) within pooled host genera (rows) from all six localities. Stars indicate idiosyncratic host and parasite species. Letters within the matrix are as in Fig. 3. July 2006 ECOLOGICAL FITTING OF ANURAN PARASITES S83

in plethodontid salamanders and has a terrestrial life cycle. Not surprisingly it is more common in a terrestrial nonranid (Pseudacris brimleyi) than in a ranid host. It is clear that a role for coevolutionary processes exists for variations in associations of parasite genera within families, and species within those genera in terms of their specific host association. However, no explanatory power is gained from considering anurans by their genera, as opposed to their ecotype, in terms of associations with the genera and families of the trematodes infecting them. This equivalence occurs, in part, because ecotype preference for anuran hosts (e.g., all aquatic and semiaquatic host species in all six sites are members of the same genus, Rana), and transmission dynamics for the parasites (e.g., all species of Haema- toleochus utilize odonate naiads as second intermediate hosts) are phylogenetically conservative (Snyder and FIG. 5. Maximally packed presence/absence matrix for trematodes (columns) pooled by genera, or by family for those Janovy 1994, Wetzel and Esch 1996). genera that did not occur in all three regions (United States, Mexico, and Costa Rica), within pooled host genera (rows) The host landscape from all six localities. Stars indicate idiosyncratic host and As evidence for phylogenetic conservatism in host and parasite species. Letters within the matrix are as in Fig. 3. parasite biology, 80% of the parasite species discovered in these six communities inhabit only 13% of the frog association between C. intermedius and Bufo marinus species sampled. How do these species coexist? Part of involves more specialization than ecological fitting. the answer lies in perhaps the most fundamental element The remaining parasite taxa, whose associations with of ecological fitting: allopatry. Only 38% of the 48 their hosts cannot easily be interpreted as manifestations parasite species inhabiting aquatic and semiaquatic of ecological fitting (based on the idiosyncratic patterns anurans occur in more than one community. revealed in the nested subset analysis), also infect Another aspect of the process of co-ocurrence lies in tadpoles at some stage in their lives. The monogeneans parasite microhabitat diversification, or, as commonly Polystoma naevius, and the probable sister species Neodiplorchis scaphiopi and Pseudiplorchis americanus, infect tadpoles and develop into adults when the tadpoles metamorphose. Anecdotal reports exist of tadpoles eating proglottids of nematotaeniid cestodes, suggesting that four additional species (Cylindrotaenia americana and C. sp., Distoichometra bufonis and D. kozloffi) gain infection in a manner similar to the first three species. Perhaps infection of a tadpole requires greater specificity on the part of the parasite than infections of adult anurans, making parasites with such life cycle patterns less amenable to ecological fitting. Habitat use by hosts Forty-two of the 58 species of adult platyhelminths (72%) occur in ranids. Of those 42 species, 27 are found only in ranids, indicating that some character or suite of characters associated with being a ranid is the resource being tracked by the parasites. Of the remaining 15 FIG. 6. Maximally packed presence/absence matrix for species, 11 always occur in higher prevalences in ranids trematodes (columns) pooled by genera, or by family for those (one measure of the host preference hierarchy), two genera that did not occur in all three regions (United States, Mexico, and Costa Rica), within host species from all six occur at lower prevalences, and two are equivocal localities, pooled by ecotype (ranked association with aquatic (possibly an artifact of small sample size). The low- habitats). Stars indicate idiosyncratic host and parasite species. prevalence occurrences in nonranid hosts might be an Variation in rank within genera was (no. species at rank) as additional example of ecological fitting if the nonranids follows: ranids, two at rank 7, six at rank 6, and two at rank 5; bufonids, two at rank 4, three at rank 5; leptodactylids, two at are suitable hosts, but their lack of exposure to aquatic rank 4, one at rank 5; hylids, two at rank 3; see Materials and habitats renders them ‘‘not apparent’’ to the parasites. methods for rank designations. No trematodes were found to Brachycoelium hospitale, for example, is generally found infect the fossorial species. S84 DANIEL R. BROOKS ET AL. Ecology Special Issue phrased, diversity in preferred site of infection within the parasite communities will be macroevolutionary mosaics host (Brooks and McLennan 1993, 2002, Adamson and of ecological fitting and co-speciation, just as are Caira 1994, Radtke et al. 2002). Platyhelminth species communities of free-living organisms (e.g., colubrid occur in the buccal cavity, lungs, gall bladder and hepatic snakes; Cadle and Greene 1993). Additionally, because ducts, small intestine, rectum, and urinary bladder of communities and associations are subject to evolu- anuran hosts, such that multiple species from different tionary forces that will vary across space and time, we clades can infect the same host and form complicated also expect that the importance of ecological fitting and communities without interacting physically, i.e., they are co-speciation will vary among communities and among microallopatric. Many species having similar trans- associations. mission modes occur in the same hosts, but live in Finally, our analysis implies that many parasites different parts of that host. On the other hand, because currently restricted to particular hosts in particular the diversification of infection site is phylogenetically localities could survive in other hosts and other localities conservative, multiple, distantly related parasite species if they could get there. Episodes of major climate change, living within a given geographic area can exhibit the for example, result in range contractions and expansions same kind of site specificity, which should amplify bringing together species that have been allopatric competition. In some cases, the parasite species occur during their previous evolutionary histories. In such in different host species; for example, polystome mono- cases, we would expect an increase in host switching, not geneans and gorgoderid digeneans live in the urinary as a result of evolution of novel host utilization bladder, but do not infect the same species of frogs. In capabilities, but as a manifestation of ecological fitting. other cases, the parasites have markedly different As discussed above (see the Introduction), some parasites biological requirements. Cestodes living in the host gut might well survive extinction of their original hosts. absorb nutrients from the host intestinal contents, Discovering the importance of ecological fitting as a whereas digeneans living in the host gut forage for host determinant of the structure of anuran parasite com- epithelial cells, cell and tissue exudates, and blood. munities thus underscores the need for more comprehensive ecological and evolutionary understanding of CONCLUSIONS host specificity in assessing the risk of parasite trans- These six communities of frog parasites are both mission into native hosts resulting from the introduction complex and similar to each other. Their complexity of exotic host species along with their parasites. rules out simple phylogenetic replication, namely, that ACKNOWLEDGMENTS these communities are products of a simple history of vicariance and/or co-speciation. The taxonomic similar- We express our appreciation to Cam Webb for inviting us to contribute to this issue. D. R. Brooks thanks the scientific and ity of the communities, coupled with their occurrence in technical staff of the Area de Conservacioń Guanacaste for such markedly different environments, rules out the support of this study, in particular: Elda Araya, Roger Blanco, possibility that they are the result of convergent Carolina Cano, Maria Marta Chavarrıá, Felipe Chavarrıá, adaptation. Brooks (1980, 1985), Futuyma and Slatkin Roberto Espinoza, Dunia Garcia, Guillermo Jimenez, Elba (1983), and Janzen (1985) suggested that relatively weak Lopez, Sigifredo Marin, Alejandro Masis, Calixto Moraga, Fredy Quesada, and Petrona Rios. Thanks to Dan Janzen and phenomena (in this case, phylogenetic conservatism in Winnie Hallwachs, scientific advisers to the ACG, for their host and parasite natural history) have the potential to support and insights. D. R. Brooks and D. A. McLennan produce marked ecological structure. That a great deal acknowledge support from the Natural Sciences and Engineer- of the stable and predictable structure of contempora- ing Research Council (NSERC) of Canada. V. Leoń-Re` gagnon ´ neous anuran parasite communities appears to be a thanks Ma. Antonieta Arizmendi, Luis Garcıa, Rosario Mata, Laura Paredes, Elizabeth Martıńez, Agustıń Jimeńez, Rogelio result of phylogenetic conservatism in the evolution of Rosas, Ulises Razo (Instituto de Biologıá, Universidad both parasite and host biology, coupled with the Nacional Autońoma de Me´xico [UNAM]), Edmundo Pe´rez, historical biogeographic contingencies of speciation Adriań Nieto, (Facultad de Ciencias, UNAM), David Lazcano, and dispersal, is consistent with those views. and Javier Banda (Universidad Autońoma de Nuevo Leoń) for their help in the field and lab, and acknowledges support from These observations, of course, do not rule out the NSF grant DEB-0102383. possibility of ongoing strong evolutionary interactions between any of these parasites and their hosts or each LITERATURE CITED other, particularly at the small spatial and short Adamson, M. L., and J. N. Caira. 1994. Evolutionary factors temporal scales associated with Thompson’s (1994) influencing the nature of parasite specificity. Parasitology coevolutionary mosaic. Nor do the observations imply 109:S85–S95. Andreń, H. 1994. Can one use nested subset pattern to reject that ecological fitting explains everything; only that the random sample hypothesis? Examples from boreal bird assumptions about the low probability of host switching communities. Oikos 70:489–491. must be viewed with far more caution in the future. Atmar, W., and B. D. Patterson. 1993. The measure of order Tracking a plesiomorphic resource in parasites is the and disorder in the distribution of species in fragmented habitat. Oecologia 96:373–382. equivalent of free-living organisms dispersing into new Atmar, W., and B. D. Patterson. 1995. The nestedness habitat, but retaining their ecological niche; both are temperature calculator: a Visual Basic program, including aspects of ecological fitting. 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APPENDIX Geographic distributions of anuran hosts and their platyhelminth parasites in six areas in North and Central America (Ecological Archives E087-112-A1). Ecology, 87(7) Supplement, 2006, pp. S86–S99 Ó 2006 by the Ecological Society of America

THE PHYLOGENETIC STRUCTURE OF A NEOTROPICAL FOREST TREE COMMUNITY

1,3 2 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL 1Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G2E9 Canada 2Department of Plant Biology, University of Georgia, Athens, Georgia 30602 USA

Abstract. Numerous ecological and evolutionary processes are thought to play a role in maintaining the high plant species diversity of tropical forests. An understanding of the phylogenetic structure of an ecological community can provide insights into the relative importance of different processes structuring that community. The objectives of this study were to measure the phylogenetic structure of Neotropical forest tree communities in the Forest Dynamics Plot (FDP) on Barro Colorado Island, Panama, to determine how the phylogenetic structure of tree communities varied among spatial scales and habitats within the FDP, and to study the effects of null-model choice on estimates of community phylogenetic structure. We measured community phylogenetic structure for tree species occurring together in quadrats ranging in size from 10 3 10 m to 100 3 100 m in the FDP. We estimated phylogenetic structure by comparing observed phylogenetic distances among species to the distribution of phylogenetic distances for null communities generated using two different null models. A null model that did not maintain observed species occurrence frequencies tended to find nonrandom community phylogenetic structure, even for random data. Using a null model that maintained observed species frequencies in null communities, the average phylogenetic structure of tree communities in the FDP was close to random at all spatial scales examined, but more quadrats than expected contained species that were phylogenetically clustered or overdispersed, and phylogenetic structure varied among habitats. In young forests and plateau habitats, communities were phylogenetically clustered, meaning that trees were more closely related to their neighbors than expected, while communities in swamp and slope habitats were phylogenetically overdispersed, meaning that trees were more distantly related to their neighbors than expected. Phylogenetic clustering suggests the importance of environmental filtering of phylogenetically conserved traits in young forests and plateau habitats, but the phylogenetic overdispersion observed in other habitats has several possible explanations, including variation in the strength of ecological processes among habitats or the phylogenetic history of niches, traits, and habitat associations. Future studies will need to include information on species traits in order to explain the variation in phylogenetic structure among habitats in tropical forests. Key words: community phylogenetic structure; environmental filtering; Forest Dynamics Plot; null models; Panama; phylogenetic clustering; phylogenetic overdispersion.

INTRODUCTION habitat specialization in tropical tree species (Hubbell Why are there so many species of trees in the tropics? and Foster 1983, Condit et al. 1996, Clark et al. 1998, Webb and Peart 2000, Harms et al. 2001), but the Tropical forests are incredibly biologically diverse, and strength of habitat specialization is often not sufficient numerous ecological and evolutionary processes, such as to explain observed levels of species richness in tropical niche differentiation, herbivory, dispersal, competition, forests (Webb and Peart 2000, Harms et al. 2001). Sim- parasitism, and disease, appear to interact to play a role ilarly, finding niche differentiation among species does in maintaining the high species diversity of tropical tree not mean that niche differences are more important than communities at a range of spatial scales (Wright 2002). species-neutral processes in structuring a community Although all of these processes have been demonstrated (Hubbell 2001). The phylogenetic structure of ecological to occur, their relative importance in structuring communities should provide insights into the relative ecological communities is not well understood. Numer- importance of different ecological processes, as these ous studies have demonstrated niche differentiation and processes interact with the evolutionary history of plant traits and leave their signature on the phylogenetic Manuscript received 21 January 2005; revised 6 September structure of a community (Webb et al. 2002). 2005; accepted 8 September 2005. Corresponding Editor (ad hoc): J. B. Losos. For reprints of this Special Issue, see footnote Previous studies have identified two general types of 1, p. S1. processes that can interact with phylogenetic patterns of 3 E-mail: [email protected] niche and trait evolution to give rise to nonrandom S86 July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S87 phylogenetic community structure, namely, competitive In this study, we used large data sets on the exclusion and environmental filtering processes (Webb phylogenetic relationships and co-occurrence patterns 2000, Webb et al. 2002, Cavender-Bares et al. 2004). of Neotropical forest tree species in communities at a Competitive exclusion and other negative density- range of local spatial scales (10 3 10 m to 100 3 100 m) dependent interactions among ecologically similar spe- to address several questions. First, we compared the cies can create nonrandom community phylogenetic performance of different null models used to study the structures. Many processes, such as herbivory (Novotny phylogenetic structure of ecological communities. Sec- et al. 2002) and competition (Uriarte et al. 2004), can ond, to test the relative importance of different have negative density-dependent effects, not only on ecological processes in structuring these communities conspecific individuals, but on close phylogenetic rela- and to test whether community phylogenetic structure tives as well. Density-dependent processes that negatively changes with spatial scale, we asked whether trees affect close phylogenetic relatives could give rise to occurring together in communities at a range of local phylogenetic overdispersion of co-occurring species, spatial scales are phylogenetically clustered or over- meaning that co-occurring species are more distantly dispersed. Third, we asked whether the phylogenetic phylogenetically related than expected by chance. Direct structure of tree communities differs among habitats competitive exclusion, as well as indirect interactions characterized by different environmental conditions. among relatives mediated by herbivores, parasites, or METHODS pathogens, could all give rise to the same pattern of community phylogenetic overdispersion (Webb et al. 2002). Ecological data Environmental filters (Weiher and Keddy 1995, Webb We estimated the phylogenetic structure of a Neo- et al. 2002, Cavender-Bares et al. 2004) or assembly rules tropical forest tree community using data from the 50-ha (Weiher and Keddy 1999) restricting community mem- Forest Dynamics Plot (FDP) on Barro Colorado Island bership to individuals possessing certain traits might in the Republic of Panama. The moist lowland forests in also affect community phylogenetic structure. If envi- the 1000 3 500 m FDP receive ;2600 mm of rain each ronmental conditions in a habitat act as a filter that year, with a dry season during January–April, and mean select for species in possession of certain traits, we would annual temperatures of 278C (Dietrich et al. 1982). expect to find either phylogenetic clustering or over- Within the FDP, a variety of habitats have been dispersion, depending on the evolutionary history of identified (Harms et al. 2001), including (in approximate ecologically important traits and species niches (Cav- order of decreasing water availability during the dry ender-Bares et al. 2004, Ackerly et al. 2006, Cavender- season) swamp, stream, slope, and upland plateaus. The Bares et al. 2006, Silvertown et al. 2006). majority of the FDP contains old-growth primary The phylogenetic structure of a community might also forests, although some relatively young secondary forest be random. If species-neutral interactions (Hubbell 2001) habitat is found within the plot (Harms et al. 2001). structure a community, if the strength of density-depend- Within the 10003500 m FDP, all tree and shrub stems ent and environmental filtering processes are balanced or 1 cm dbh have been mapped and identified to species in weak, or if species niches or traits are phylogenetically repeated censuses conducted since 1982 (Condit 1998). random, local communities could exhibit phylogenetic We measured the phylogenetic structure of the forests on structures indistinguishable from random. Barro Colorado Island using data on occurrence of tree Given the variation of measures of community and shrub species in the FDP. Mapped tree locations structure, such as taxonomic and functional diversity from the 1982 census of the FDP were divided into square with spatial scale and extent (Levin 1992), and changes in nonoverlapping quadrats of four different spatial scales the phylogenetic signal of niches and traits with spatial (10 3 10 m, 20 3 20 m, 50 3 50 m, and 100 3 100 m). We scale (Cavender-Bares et al. 2006, Silvertown et al. 2006), defined communities at a given spatial scale to include we would predict that the phylogenetic structure of a species of all tree and shrub stems 1 cm dbh present community will be highly dependent on the spatial scale together in individual quadrats at that scale. Although and extent used to define the community. werefertothecommunitiesintheFDPastree The choice of an appropriate null model to use when communities throughout this paper for the sake of measuring the structure of ecological communities has convenience, shrubs 1 cm dbh were also included in been very contentious (Gotelli and Graves 1996, Gotelli all analyses of community structure. 2004), since analyses of the same data set with different null models can lead to very different conclusions. Simu- Phylogenetic data lation studies have been used to directly assess the perfor- We constructed a hypothesized phylogenetic tree for mance of different null models when measuring species the 312 tree species occurring in the FDP using co-occurrence patterns (e.g., Gotelli 2000), and a number Phylomatic version R20031202 software (Webb and of different null models have been used to measure com- Donoghue 2005), a phylogenetic database and toolkit munity phylogenetic structure, but the effects of different for the assembly of phylogenetic trees. The tree created by null models on estimates of community phylogenetic Phylomatic used information from numerous published structure have not been evaluated quantitatively. molecular phylogenies to create a tree containing all of S88 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL Ecology Special Issue

FIG. 1. Hypothesized phylogenetic relationships among woody plant species occurring in the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama. Circles indicate nodes dated based on divergence dates reported by Wikstro¨m et al (2001). Undated nodes were spaced evenly between dated nodes to minimize variation in branch length. the species in the FDP, based on Phylomatic reference All analyses of community phylogenetic structure were tree R20031202 with the APG II (Angiosperm Phylogeny conducted using the Phylocom version 3.19 software Group 2003) phylogenetic classification of flowering package (Webb et al. 2004). Three steps were taken to plant families (P. F. Stevens, available online)4 forming measure community phylogenetic structure in each the backbone of the tree. In the absence of detailed quadrat. First, we calculated raw phylogenetic distances information on phylogenetic relationships within many among species occurring together in each quadrat. of the 55 families and 192 genera found in the FDP, we Second, we created numerous randomly generated null assumed most families and genera were monophyletic communities corresponding to each quadrat and esti- and polytomous when placing them on the tree. We mated raw phylogenetic distances among species occur- assigned branch lengths to the phylogenetic tree using the ring together in the null communities. Finally, we BLADJ module of the Phylocom version 3.19 software calculated measures of standardized effect size (Gotelli package (Webb et al. 2004), creating a pseudochrono- and Rohde 2002) of community phylogenetic structure gram with branch lengths based on clade ages reported by for our raw measures of phylogenetic distance in each Wikstro¨m et al. (2001). Nodes in the phylogenetic tree for quadrat, by comparing observed phylogenetic distances which age estimates were available were fixed at their to the distribution of phylogenetic distances for the estimated ages (Wikstro¨m et al. 2001), and all remaining randomly generated null communities. branch lengths were set by spacing undated nodes in the We calculated raw phylogenetic distances among tree evenly between dated nodes to minimize variance in species in quadrats in two ways, each of which captures branch lengths. Age estimates were available for 34 of the a different aspect of the phylogenetic relatedness of co- 122 internal nodes in the phylogeny. Although 57 of the occurring species (Webb 2000). The mean pairwise 122 internal nodes were polytomous, the majority of distance (MPD) was calculated as the mean phylogenetic these polytomies were within families and genera, with distance among all pairwise combinations of species occurring together in each quadrat, and the mean the backbone of the tree relatively well resolved and nearest neighbor distance (MNND) was calculated as dated. The resulting phylogenetic tree (Fig. 1; also, see the the mean phylogenetic distance to the nearest relative Supplement) was used for all subsequent analyses of for all species occurring together in each quadrat (Webb community phylogenetic structure. 2000, Webb et al. 2002). Community phylogenetic structure Null models We calculated several measures of community phylo- To determine whether the phylogenetic structure of genetic structure for all quadrats at each spatial scale. local tree communities differed from the phylogenetic community structure expected by chance, we compared 4 hhttp://www.mobot.org/MOBOT/research/APwebi observed phylogenetic distances among species in each July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S89 quadrat to the distribution of phylogenetic distances for species occurring in each quadrat, relative to the randomly generated null communities (Gotelli and distribution of distances calculated for null communities Graves 1996). To assess the effect of null-model choice in each quadrat. These effect size measures compared on ability to measure community phylogenetic structure, the observed phylogenetic distance in each quadrat to we generated null communities in two ways. the distribution of phylogenetic distances in null The first method maintained the total species richness communities corresponding to that quadrat, and can of each quadrat, with species in each quadrat chosen be used to test for phylogenetic clustering or over- equiprobably at random, without replacement from the dispersion. We used two metrics of community phylo- pool of species present in the FDP. We refer to this null genetic structure similar to those first proposed by Webb model as the ‘‘unconstrained’’ model, since the species (2000), but based on comparisons with different null richness of each quadrat remained the same in the null models. communities, but species occurrence frequencies in the The net relatedness index (NRI) of each quadrat null communities were not constrained to be equal to (Webb 2000, Webb et al. 2002) was defined as [–(MPD – their actual occurrence frequency among quadrats in the MPDnull)/SD(MPDnull)], where MPD is the mean pair- FDP data set. This null model assumes that all species wise phylogenetic distance among species in the quadrat, present in the FDP are equally able to colonize any MPDnull is the mean MPD for that quadrat in 1000 null quadrat. communities, and SD(MPDnull) is the standard deviation The second method maintained both the total species of MPD for that quadrat in 1000 null communities. The richness of each quadrat, as well as the occurrence net relatedness index has been proposed as a measure of frequency of each species, by randomly swapping species tree-wide phylogenetic clustering and overdispersion of occurrences among all quadrats at a scale subject to the species (Webb 2000). Positive NRI scores indicate that constraint that the species richness of each quadrat species occurring together in a quadrat are more closely remain constant and that the relative frequency of all phylogenetically relatedthanexpectedbychance, species occurrences in quadrats remain constant. We generally due to tree-wide phylogenetic clustering of refer to this null model as the ‘‘constrained’’ model, since co-occurring species. Negative NRI scores indicate that the occurrence frequencies of species in the null co-occurring species are less phylogenetically related community were constrained to be equal to their actual than expected by chance, generally due to tree-wide frequency in quadrats at that spatial scale. This null phylogenetic overdispersion of co-occurring species. model assumes that a species’ ability to colonize a The nearest taxon index (NTI) of each quadrat (Webb quadrat is proportional to its frequency in the FDP. et al. 2002) was defined as [–(MNND – MNNDnull)/ We generated the constrained null communities using SD(MNNDnull)], where MNND is the mean nearest the independent swap algorithm (Gotelli 2000, Gotelli neighbor phylogenetic distance among species in the and Entsminger 2003) by holding the row and column quadrat, MNNDnull is the mean MNND for that sums of the quadrat/species occurrence matrix constant quadrat in 1000 null communities, and SD(MNNDnull) while swapping species among quadrats using a checker- is the standard deviation of MNND for that quadrat in board swap. The checkerboard swap searches the 1000 null communities. The nearest taxon index has quadrat/species matrix for submatrices of the form been proposed as a measure of terminal (branch tip) (0,1)(1,0) or (1,0)(0,1) (where 1 and 0 represent species phylogenetic clustering of species on a phylogeny (Webb presence and absence, respectively, in two quadrats) and 2000). If species tend to occur together with other closely swaps species presences between quadrats when these related species (e.g., with congeners or confamilials), checkerboard submatrices are found. This maintains NTI scores will generally be positive due to this terminal species frequencies and quadrat species richnesses while phylogenetic clustering of species toward the tips of the randomizing patterns of species co-occurrence. Each phylogenetic tree. If species tend not to occur together null community was created by swapping subsequent with other closely related species, NTI scores will be matrices many times relative to the number of species negative due to terminal phylogenetic overdispersion. presences in the quadrat/species matrix, creating serially independent randomized matrices. The first null com- Estimating plot-wide phylogenetic structure munity for each spatial scale was created by checker- To test whether the average phylogenetic structure of board swapping the original quadrat/species matrix for local tree communities at a given spatial scale differed that scale 30 000 times, with each subsequent null from random, we calculated the mean phylogenetic community generated by checkerboard swapping the structure of all quadrats at each scale as the mean NRI previous matrix 10 000 times. and NTI of all quadrats at that scale. If the mean NRI We recorded the mean and standard deviation of or NTI for all quadrats at a given spatial scale differed MPD and MNND among species in each quadrat for from zero according to a one-sample t test, we could 1000 null communities generated using the constrained conclude that the tree communities at that scale were and unconstrained null models. We then calculated significantly phylogenetically clustered or overdispersed measures of standardized effect size (Gotelli and Rohde on average, since both NRI and NTI are standardized 2002) of the observed phylogenetic distances among effect sizes whose expected values are zero for phylo- S90 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL Ecology Special Issue

FIG. 2. Spatial patterns of (a) habitats (from Harms et al. [2001]), (b) net relatedness index (NRI), and (c) nearest taxon index (NTI) in 20 3 20 m quadrats within the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama. The indices NRI and NTI are measures of community phylogenetic structure based on a constrained null model that shufﬂed species co-occurrence patterns in null communities while maintaining observed species occurrence frequencies and quadrat species richnesses (see Methods for description). Positive NRI and NTI values indicate phylogenetic clustering, and negative values indicate phylogenetic overdispersion of species occurring together in a quadrat. genetically random communities, positive for phyloge- structure for each quadrat using the constrained and netically clustered communities, and negative for phy- unconstrained null models. logenetically overdispersed communities, with We also assessed the phylogenetic structure of each approximately 95% of NRI and NTI values expected quadrat by comparing the observed MPD and MNND to fall in the range of 2toþ2 for random communities values in each quadrat to the distribution of these values (Gotelli and Rohde 2002). We estimated phylogenetic in the null communities. Quadrats were considered to be July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S91

TABLE 1. Results of a randomization study to assess the ability of different null models to measure community phylogenetic structure for 50 randomly selected 20 3 20 m quadrats in the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama.

A) Mean pairwise phylogenetic distance (MPD) and net relatedness index (NRI) MPD (Ma) Randomization Analysis NRI Randomization Analysis Type I null model null model Mean Mean SD Mean error rate Constrained constrained 214.3 214.4 5.9 0.00 0.06 unconstrained 214.3 217.9 7.3 0.49 0.04 Unconstrained constrained 217.9 217.9 7.2 0.00 0.06 unconstrained 217.9 217.9 7.2 0.00 0.06 B) Mean nearest neighbor phylogenetic distance (MNND) and nearest taxon index (NTI) MNND (Ma) Randomization Analysis NTI Randomization Analysis Type I method method Mean Mean SD Mean error rate Constrained constrained 78.0 78.1 6.6 0.02 0.05 unconstrained 78.0 79.2 7.2 0.18 0.05 Unconstrained constrained 79.2 79.2 7.1 0.00 0.06 unconstrained 79.2 79.2 7.2 0.00 0.05 Notes: Species co-occurrences in these quadrats were first randomized using either the constrained or unconstrained null model (see Methods for description). Phylogenetic distances among co-occurring species (mean pairwise phylogenetic distance [MPD] and mean nearest neighbor phylogenetic distance [MNND]) were first calculated for the randomized data. Both MPD and MNND are reported in terms of millions of years (Ma). The distributions of phylogenetic distances in null communities were then calculated for 999 subsequent randomizations of the data, using the analysis null model, and used to calculate measure of community phylogenetic structure for each randomization (net relatedness index [NRI] and nearest taxon index [NTI]; see Methods for description). This process was repeated 500 times for each combination of randomization and analysis null models. The average community phylogenetic structure (mean NRI and NTI) and Type I error rate (proportion of randomizations indicating a significant phylogenetic structure [absolute value of NRI or NTI . 2, P , 0.05]) were calculated for each combination of randomization and analysis null models. Boldface type indicates mean NRI or NTI values that were significantly different from zero according to a one-sample t test (N ¼ 50 quadrats 3 500 runs, P , 0.05). significantly phylogenetically overdispersed or clustered et al. 1998). These models calculate an estimate of the if they occurred in the lowest or highest 2.5% of the mean and standard error of the coefficients in the model distribution of distances from the null communities, (NRI or NTI values), taking into account the non- respectively (a ¼ 0.05). A one-tailed binomial test was independence of spatially adjacent samples (Cressie then used to assess whether the numbers of quadrats with 1993). In all cases, adding a first-order spatial-neighbor significantly overdispersed or clustered phylogenetic autoregressive term to the model removed autocorrela- distances were greater than expected at each spatial scale. tion from the residuals and improved the fit of the Differences in sample sizes among spatial scales were model, relative to a nonspatial model, and so we report accounted for using bootstrap estimation (Manly 1997) only the results of the spatial models. of the mean and standard error of NRI and NTI at each spatial scale. At the largest spatial scale examined (100 3 Null model comparisons 100 m), the sample size was 50 quadrats. At smaller To compare null models, we employed a method spatial scales (50 3 50 m, 20 3 20 m, 10 3 10 m), we similar to that used by Gotelli (2000), whereby random estimated the mean and standard error of phylogenetic communities are created by shuffling ‘‘real’’ data using structure (NRI and NTI) for 4999 random draws null models in order to randomize patterns of species co- without replacement of 50 quadrats. The bootstrap occurrence, and then the randomized data are analyzed estimates of mean and standard errors of NRI and NTI using the same set of null models. We randomly chose 50 were then used to calculate a bootstrap t statistic and P of the FDP’s 20 3 20 m quadrats to conduct our value for NRI and NTI at each spatial scale. This randomization study. We first randomized the original method allowed direct comparisons of the mean and data using the unconstrained or constrained null models. standard error of NRI and NTI values among spatial This created a new set of samples with the same phylo- scales, taking into account the original differences in genetic relationships among species and sample species sample size at each spatial scale. richnesses as the original data, but for which species co- Because phylogenetic similarities among co-occurring occurrences were completely randomized. We then species were positively spatially autocorrelated (Fig. 2), calculated phylogenetic distances (MPD and MNND) we also tested whether NRI and NTI values at each and standardized effect sizes of community phylogenetic spatial scale differed from zero using generalized least- structure (NRI and NTI) for the randomized samples squares models with simultaneous spatial autoregression using 1000 runs of the unconstrained and constrained (SAR) covariance structures in SþSpatialStats (Kaluzny null models. This process was repeated 500 times for S92 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL Ecology Special Issue

FIG. 3. Tree community phylogenetic structure in 20 3 20 m quadrats within the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama. Observed phylogenetic distances among co-occurring species are presented for 1250 quadrats, along with phylogenetic distances (in millions of years, Ma) among species in corresponding null communities generated using a constrained and an unconstrained null model (see Methods for description). each combination of randomization and analysis null process that acts to determine the occurrence frequency models. of a species (Colwell and Winkler 1984). If this were We estimated the proportion of randomized samples occurring, we would expect some phylogenetic signal in for which each null model found a signiﬁcantly non- the distribution of species occurrence frequencies. To random phylogenetic distance among species (number of determine whether the frequencies of species in the FDP times observed distances were in the top or bottom 2.5% exhibited a phylogenetic signal, we calculated phyloge- of randomized distances, an estimate of the Type I error netic distances among all pairs of species occurring in rate), as well as calculating the average degree of phylo- the entire FDP at each spatial scale examined. We then genetic clustering or overdispersion (mean NRI and calculated the dissimilarity of species frequencies as the NTI) estimated by each null model. square root of the absolute difference in occurrence Null models that maintain species frequencies have frequency rank for all pairs of species, and we tested the been criticized for potentially including the effects of any signiﬁcance of the correlation among the resulting July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S93

FIG. 4. Relationship between measures of phylogenetic community structure calculated using two null models in 1250 20 3 20 m quadrats within the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama. Net relatedness index (NRI) and nearest taxon index (NTI) are measures of community phylogenetic structure based on constrained and unconstrained null models (see Methods for description). phylogenetic and frequency distance matrices using a young), we asked whether the phylogenetic structure of Mantel test (Legendre and Legendre 1998). tree communities at this spatial scale differed among habitats within the FDP. We tested for overall differ- Habitat phylogenetic structure ences in phylogenetic structure among habitats using Based on Harms et al.’s (2001) classiﬁcation of 20 320 generalized least-squares models with simultaneous m quadrats within the FDP into seven habitat types (high spatial autoregression (SAR) covariance structures, as plateau, low plateau, mixed, slope, stream, swamp, and described for the plot-wide tests. We estimated the

FIG. 5. Relationship between phylogenetic distances among species and similarity of species occurrence frequency ranks in 20 3 20 m quadrats within the 50-ha Forest Dynamics Plot (FDP) on Barro Colorado Island, Panama. Data points represent all pairwise combinations of species present in the FDP. Frequency rank differences were calculated as the absolute difference in the frequency occurrence ranks of each species pair. Phylogenetic distances were calculated as the branch length (in millions of years, Ma) connecting each species pair. Mantel tests indicated that relationships between square-root transformed frequency rank difference and phylogenetic distance were not statistically signiﬁcant at any spatial scale examined. S94 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL Ecology Special Issue

TABLE 2. Tree community phylogenetic structure in quadrats at four spatial scales within the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama.

Net relatedness index Nearest taxon index

Spatial scale N Estimated mean SE P Estimated mean SE P Unconstrained null model 10 3 10 m 5000 0.260 0.008 0.0001 0.142 0.012 0.0001 20 3 20 m 1250 0.389 0.016 0.0001 0.154 0.021 0.0001 50 3 50 m 200 0.771 0.047 0.0001 0.453 0.045 0.0001 100 3 100 m 50 0.074 0.099 0.4605 0.019 0.099 0.8459 Constrained null model 10 3 10 m 5000 0.061 0.016 0.0001 0.070 0.014 0.0001 20 3 20 m 1250 0.056 0.033 0.0952 0.020 0.028 0.4854 50 3 50 m 200 0.051 0.093 0.5825 0.046 0.076 0.5427 100 3 100 m 50 0.043 0.180 0.8110 0.003 0.161 0.9858 Notes: Net relatedness index (NRI) and nearest taxon index (NTI) are measures of community phylogenetic structure based on constrained and unconstrained null models (see Methods for description). Positive NRI and NTI values indicate phylogenetic clustering; negative values indicate phylogenetic overdispersion of species occurring together in a quadrat. Parameter estimates at each scale are based on a spatial generalized least-squares model with a first-order spatial-neighbor simultaneous spatial autoregression term (SAR). Significant P values indicate that the phylogenetic structure at a given spatial scale differed from zero according to a two-tailed t test. overall significance of differences in phylogenetic struc- and NTI values calculated for each quadrat using the ture among habitats using NRI and NTI scores based on different null models were tightly correlated (Fig. 4), but the constrained null model, as well as estimating the NRI and NTI values calculated using the unconstrained mean and standard errors of NRI and NTI scores within null model tended to be higher than those calculated each habitat type to test for significant phylogenetic using the unconstrained null model. clustering or overdispersion. In all cases, adding a first- We found no statistically significant relationships order spatial-neighbor autoregressive term to the model between phylogenetic distances among species and the removed autocorrelation from the residuals and im- square root of differences in species frequency ranks at proved the fit of the model relative to a nonspatial model, any spatial scale (Mantel tests, P . 0.5 at all scales), and so we report only the results of the spatial models. although there was a slight but nonsignificant trend of the most closely related species pairs having similar RESULTS frequency ranks (Fig. 5). Null model comparisons Community phylogenetic structure When analyzing community data generated by The phylogenetic structure of tree communities in the randomizing 50 randomly selected 20 3 20 m quadrats FDP was highly variable among quadrats and depend- from the Forest Dynamics Plot with the unconstrained ent on the choice of null model. Using the unconstrained null model, both null models performed well (Table 1), null model, tree communities in the FDP were phylo- with appropriate Type I error rates and mean NRI and genetically clustered on average (mean NRI and NTI . NTI values of approximately zero. When analyzing data 0; Table 2, Fig. 6) at spatial scales from 10 3 10 m to 50 generated by randomizing these quadrats with the 3 50 m. Although the mean NRI and NTI were greater constrained null model, the unconstrained null model than zero at most scales examined, relatively few concluded that the randomized data were significantly quadrats were significantly phylogenetically clustered phylogenetically clustered or overdispersed (mean NRI or overdispersed according to the unconstrained null and NTI different from zero), although the Type I error model (Table 3, Fig. 6). rates of both null models remained correct (Table 1). According to the constrained null model, mean A comparison of the phylogenetic distances among community phylogenetic structure (NRI and NTI) co-occurring species in all 1250 20 3 20 m quadrats in across the entire plot did not differ from zero, except the FDP with the corresponding mean pairwise phylo- at the smallest spatial scale examined (10 3 10 m), where genetic distances in null communities (Fig. 3) showed the mean NRI was greater than zero, indicating a slight that phylogenetic distances in the null communities were overall trend of phylogenetic clustering at this scale. The much less variable than the observed distances, espe- magnitude of this effect was very small, and, after cially the mean pairwise distances. Mean phylogenetic accounting for differences in sample size among spatial distances among co-occurring species in the uncon- scales using bootstrap resampling (Table 4), the average strained null communities were higher than the mean phylogenetic structure (NRI and NTI) of tree commun- distances in the observed and constrained null communities did not differ from zero at all spatial scales ities. As a result of the higher mean phylogenetic examined according to the constrained null model. distances in the unconstrained null communities, NRI However, there was substantial variation in phylogenetic July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S95

FIG. 6. Tree community phylogenetic structure in quadrats at four spatial scales within the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama. See Fig. 4 legend for definition and description of (a) NRI and (b) NTI. Dashed lines indicate expected 95% CI. structure around the mean at all spatial scales examined ficant positive spatial autocorrelation of NRI and NTI (Fig. 6), and more quadrats than expected contained values and nonsignificant spatial autocorrelation of re- communities that were significantly phylogenetically siduals from the spatial models were observed at all overdispersed or clustered (Table 1). spatial scales examined. In the 20 3 20 m quadrats (Fig. 2), the phylogenetic Habitat influences on community phylogenetic structure structure of a quadrat was positively correlated with the phylogenetic structures of that quadrat’s first-order Based on the constrained null model, the phylogenetic spatial neighbors (NRI, Moran’s I ¼ 0.34, P , 0.01; structure of the tree communities in the FDP differed NTI, Moran’s I ¼ 0.14, P , 0.01), whereas the residuals among habitats at the spatial scale (20 3 20 m) for which of the spatial autoregression models for these values habitat data were available (Fig. 1, Table 5). Species were not significantly spatially correlated (NRI resid- occurring together in the high plateau, low plateau, and uals, Moran’s I ¼0.05, P ¼ 0.93; NTI residuals, young habitats tended to be significantly phylogeneti- Moran’s I ¼0.02, P ¼ 0.80). Similar patterns of signi- cally clustered (NRI . 0 or NTI . 0, P , 0.05), while S96 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL Ecology Special Issue

TABLE 3. Number of quadrats with statistically signiﬁcant phylogenetic clustering or overdispersion at four spatial scales in the 50- ha Forest Dynamics Plot on Barro Colorado Island, Panama.

MPD MNND Total Overdispersed quadrats Clustered quadrats Overdispersed quadrats Clustered quadrats quadrats Spatial scale NNBinomial PNBinomial PN Binomial PNBinomial P Unconstrained null model 10 3 10 m 5000 69 1.0000 4 1.0000 92 0.9992 17 1.0000 20 3 20 m 1250 36 0.2171 1 1.0000 10 1.0000 0 1.0000 50 3 50 m 200 17 ,0.001 1 0.9937 1 0.9937 0 1.0000 100 3 100 m 50 0 1.0000 3 0.1294 0 1.0000 1 0.7180 Constrained null model 10 3 10 m 5000 188 ,0.001 204 ,0.001 150 0.0151 98 0.9950 20 3 20 m 1250 76 ,0.001 63 ,0.001 30 0.6144 40 0.0716 50 3 50 m 200 18 ,0.001 6 0.3840 4 0.7385 6 0.3840 100 3 100 m 50 3 0.1294 1 0.7180 4 0.0362 2 0.3565 Notes: Phylogenetic distances among co-occurring species in each quadrat (mean pairwise phylogenetic distance [MPD] and mean nearest neighbor phylogenetic distance [MNND]) were compared with phylogenetic distances in 1000 null communities generated using the constrained and unconstrained null models (see Methods for description). Quadrats were considered to be significantly phylogenetically overdispersed or clustered if they occurred in the lowest or highest 2.5% of the distribution of distances from the null communities, respectively (a ¼ 0.05). A one-tailed binomial test was then used to assess whether the numbers of quadrats with significantly overdispersed or clustered phylogenetic distances were greater than expected at each spatial scale. communities in the swamp habitat tended to contain species, but also their occurrence frequencies across species that were phylogenetically overdispersed (NRI ¼ samples and the distribution of frequencies on the 1.096, P , 0.0001). Communities in the slope habitat phylogeny. exhibited both tree-wide phylogenetic overdispersion Randomization tests showed that the unconstrained (NRI ¼0.286, P ¼ 0.0005) and marginally significant null model can indicate nonrandom community phylo- terminal phylogenetic clustering (NTI ¼ 0.130, P ¼ genetic structure (mean NRI and NTI different from 0.0589). zero) when used with data containing nonuniform species frequencies, even when patterns of species co- DISCUSSION occurrence in samples are completely random (Table 1). Null model choice and community phylogenetic structure A similar pattern was found in the tree communities Measures of phylogenetic distances among species in a within the FDP (Fig. 3). In the unconstrained null community must be compared with the phylogenetic communities, mean pairwise phylogenetic distance con- distances generated by a null model in order to deter- verged on the average pairwise phylogenetic distance mine whether a community is more phylogenetically among all species occurring in the FDP, with every clustered or overdispersed than expected by chance. A species and its associated phylogenetic distance to other potential problem with analyses of the phylogenetic species given equal weighting. In the constrained null structure of a community is that null models can simul- communities, species and associated phylogenetic taneously affect not only the co-occurrence patterns of branches were effectively weighted by their frequency

TABLE 4. Tree community phylogenetic structure in quadrats at four spatial scales within the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama.

Net relatedness index Nearest taxon index

Spatial scale N Estimated mean SE P Estimated mean SE P Unconstrained null model 10 3 10 m 50 0.261 0.077 0.0013 0.143 0.118 0.2327 20 3 20 m 50 0.388 0.079 ,0.0001 0.156 0.103 0.1347 50 3 50 m 50 0.771 0.094 ,0.0001 0.452 0.090 ,0.0001 100 3 100 m 50 0.074 0.099 0.4583 0.019 0.099 0.8455 Constrained null model 10 3 10 m 50 0.058 0.156 0.7128 0.070 0.140 0.6201 20 3 20 m 50 0.053 0.157 0.7389 0.021 0.139 0.8786 50 3 50 m 50 0.047 0.186 0.8028 0.047 0.151 0.7546 100 3 100 m 50 0.043 0.180 0.8122 0.003 0.161 0.9852 Notes: Parameter estimates at each scale are based on 4999 bootstrap resamples of 50 quadrats from that spatial scale, except at the 100 3 100 m scale where parameters represent the actual parameter estimates for the 50 quadrats at that scale. Signiﬁcant P values indicate that the phylogenetic structure at a given spatial scale differed from zero according to a two-tailed t test. See Table 2 notes for additional deﬁnitions and explanations. July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S97

TABLE 5. Tree community phylogenetic structure in 20 3 20 m quadrats in different habitats within the 50-h Forest Dynamics Plot on Barro Colorado Island, Panama.

Net relatedness index Nearest taxon index

Habitat N Mean SE P Mean SE P High Plateau 170 0.338 0.117 0.0039 0.021 0.094 0.8216 Low Plateau 620 0.117 0.060 0.0533 0.054 0.049 0.2619 Mixed 66 0.140 0.146 0.3393 0.141 0.129 0.2723 Slope 284 0.286 0.827 0.0005 0.130 0.069 0.0589 Stream 32 0.198 0.219 0.3666 0.315 0.191 0.0990 Swamp 30 1.096 0.244 0.0001 0.116 0.206 0.5720 Young 48 0.570 0.206 0.0057 0.440 0.169 0.0095 Notes: Net relatedness index (NRI) and nearest taxon index (NTI) are measures of community phylogenetic structure based on a constrained null model that shuffled species co-occurrence patterns in null communities while maintaining observed species occurrence frequencies and quadrat species richnesses (see Methods for description). Parameter estimates in each habitat are based on a spatial generalized least-squares (GLS) model with a first-order spatial neighbor simultaneous spatial autoregression term (SAR). Overall differences among habitats in NRI and NTI were statistically significant according to the spatial GLS tests (P , 0.0001). Significant P values in the table indicate that the phylogenetic structure in a habitat differed from zero (significant phylogenetic clustering or overdispersion) according to a two-tailed t test. See Table 2 notes for additional definitions and explanations. within the plot, leading to similar mean phylogenetic (Wilson 1995). Conversely, null models that maintain distances in the observed and constrained null commun- species frequencies have been criticized for potentially ities. ‘‘smuggling in’’ the effects of processes such as competi- Our results highlight the sensitivity of measures of tion or environmental filtering on species frequencies phylogenetic community structure, not only to patterns and community phylogenetic structure (the ‘‘Narcissus of species co-occurrence, but also to phylogenetic tree effect’’ [Colwell and Winkler 1984]), and for potentially topology, branch lengths, and species frequencies. By being too statistically conservative. If this were the case, giving species such as the tree fern Cnemidaria petiolaria we might expect some relationship between species’ (which is both rare and distantly related to all other frequencies and their phylogenetic relatedness, but species in the FDP) an equal chance of occurring in relationships between frequency similarity and phyloge- every null community, phylogenetic distances among netic distance were not statistically significant at any species in the unconstrained null communities were spatial scale examined according to Mantel tests (Fig. 2). inflated relative to the observed communities, causing We might also expect the constrained null model to find mean NRI and NTI values to be greater than zero on nonrandom community structure in fewer quadrats average. Conversely, by maintaining species frequencies within the FDP if it were too statistically conservative, in null communities, the constrained null communities but in fact the constrained model found many more tended to have the same mean phylogenetic structure as quadrats to be phylogenetically nonrandom compared the observed communities, leading to a distribution of to the unconstrained null model (Table 2), although NRI and NTI values whose mean was close to zero. mean NRI and NTI values from the constrained null When using the unconstrained null model with the model were always close to zero. FDP data, it is difficult to attribute any differences in Recent attempts to resolve the null model debate have phylogenetic structure between the observed and null focused on the use of simulation studies to quantify the communities to the impact of ecological processes on the Type I and II error rates of different null models when phylogenetic structure of the community, since they confronted with randomized or simulated community might simply be due to differences in species frequencies co-occurrence data (Gotelli 2000). Although we quanti- between the observed and null communities. Addition- fied the Type I error rate and bias of null models in this ally, given the widespread spatial aggregation of trees study, we did not assess the Type II error rate, which will (Condit et al. 2000) and strong dispersal limitation need to be measured using simulation studies with data (Hubbell et al. 1999) demonstrated to occur in the FDP generated using different models of the interaction and other ecological communities, the assumption that among phylogenetic relationships, trait evolution, and all species are equally able to colonize any sample in the community structure. null communities is not realistic. Several additional issues related to the use of null Much of the previous debate surrounding the use of models to measure community phylogenetic structure null models in ecology has focused on the relative merits remain unresolved. The constrained null model used in of null models that maintain or do not maintain species this study maintains observed species frequencies in null frequencies (Gotelli and Graves 1996). Null models that communities, but swaps species presences among sam- do not maintain species frequencies have been criticized ples, and thus can only be used with species presence/ as being overly statistically liberal; this shortcoming has absence data matrices. Species abundances within been described previously as the ‘‘Jack Horner effect’’ samples contain useful information that is discarded S98 STEVEN W. KEMBEL AND STEPHEN P. HUBBELL Ecology Special Issue when using this approach, but it is probably not possible young forests in the FDP suggests that some sort of to simultaneously constrain species frequencies, abun- environmental filter is structuring tree communities in dances, and sample species richnesses. these habitats. Habitats such as the dry plateaus within The constrained null model also ignores species in the the FDP would be predicted to contain phylogenetically regional species pool when generating null communities, clustered species, since the relatively stressful dry-season since only species present in local communities are used soil moisture conditions in these environments would be to generate the null communities. To determine how more likely to act as an environmental filter on broadly community phylogenetic structure varies across a larger conserved ecological traits than in moister habitats range of spatial scales (Webb et al. 2002, Cavender- (Harms et al. 2001, Webb et al. 2002). Bares et al. 2006), it will be necessary to compare the Communities in the relatively moist slope and swamp phylogenetic relatedness of species present in commun- habitats were phylogenetically overdispersed (Table 5). ities at one scale to those present in some regional Tree-wide phylogenetic overdispersion in the swamp species pool. It is not clear how to separate the effects of habitats was caused by the presence of species from variation in species frequency from the effects of families such as the Moraceae and Arecaceae (Harms et ecological and evolutionary processes on community al. 2001) that are widely scattered across the angiosperm phylogenetic structure for these types of data, but clearly phylogeny. In the slope habitats, a pattern of tree-wide more research on the effects of null-model and species overdispersion, but terminal phylogenetic clustering, pool choice is needed. was found. Several processes could give rise to these patterns of Community phylogenetic structure in the FDP phylogenetic overdispersion, depending on the interac- Although the average phylogenetic structure of the tion between the phylogenetic history of trait evolution tree communities in the FDP was close to random on and contemporary ecological interactions in these average across the entire plot at spatial scales from 10 3 habitats. Even within a single habitat type, the hier- 10 m to 100 3 100 m according to the constrained null archical nature of trait and niche evolution (Ackerly et model (Tables 2 and 4), the phylogenetic relatedness of al. 2006, Silvertown et al. 2006), interactions among tree species occurring together in individual quadrats multiple ecological processes (Webb et al. 2002), and the varied greatly from phylogenetic overdispersion to phy- phylogenetic history of species habitat associations logenetic clustering (Table 2, Fig. 6), and more quadrats (Brooks and McLennan 1991) could lead to compli- than expected exhibited significant phylogenetic cluster- cated patterns of community phylogenetic structure, ing and overdispersion at most spatial scales (Table 1, making it difficult to attribute these patterns to any one Fig. 6). Habitats within the plot differed strongly in process. their phylogenetic structure (Table 5, Fig. 2). At the More data on the ecological traits of species in the spatial scale for which habitat data were available, FDP and the evolutionary history of habitat associa- phylogenetic clustering in some habitats combined with tions in tropical trees will be necessary to determine the phylogenetic overdispersion in other habitats appeared relative importance of processes such as environmental to result in a pattern of phylogenetic structure filtering or competitive exclusion in different habitats indistinguishable from random on average across the within the FDP. However, it is clear that either the entire FDP, obscuring the strong differences in phylo- relative importance of nonneutral ecological processes genetic structure among habitats within the FDP. or the evolutionary history of niches, traits, or habitat In the seasonally dry plateau habitats and in the associations must vary along environmental gradients young secondary growth forests within the plot, co- within the FDP to explain the observed variation in occurring species were phylogenetically clustered (Table phylogenetic structure among habitats. 5). The tree-wide phylogenetic clustering in the plateau habitats was largely due to the co-occurrence of numer- ACKNOWLEDGMENTS ous species from large, highly speciose clades such as the Thanks to Cam Webb for inviting this contribution and for eurosids. Numerous species from these clades tended to suggesting the phylogenetic analysis of the Forest Dynamics Plot (FDP) data. Thanks to Kyle Harms for providing the occur together in plateau quadrats, leading to a trend of habitat data, and to Rick Condit for making the FDP data tree-wide phylogenetic clustering of co-occurring species available. Thanks to J. F. Cahill and M. R. T. Dale for in the plateau. Similarly, in the young habitats, species statistical advice. This manuscript benefited greatly from com- from families and genera concentrated in a few orders ments by C. Webb and three anonymous reviewers. S. W. commonly occurred together in most quadrats, leading Kembel acknowledges support from the Natural Sciences and Engineering Research Council of Canada. The Forest Dynam- to a pattern of both tree-wide and terminal phylogenetic ics Plot of Barro Colorado Island has been made possible clustering. through the generous support of the U.S. National Science Given the broad phylogenetic niche and trait con- Foundation, The John D. and Catherine T. MacArthur servatism documented across plants in general (Prinzing Foundation, and the Smithsonian Tropical Research Institute and through the hard work of over 100 people from 10 et al. 2001, Cavender-Bares et al. 2006, Silvertown et al. countries over the past two decades. The BCI Forest Dynamics 2006) and tropical trees in particular (Chazdon et al. Plot is part of the Center for Tropical Forest Science, a global 2003), the phylogenetic clustering in the plateau and network of large-scale demographic tree plots. July 2006 FOREST COMMUNITY PHYLOGENETIC STRUCTURE S99

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SUPPLEMENT A phylogenetic tree and key to species codes for tree species occurring within the 50-ha Forest Dynamics Plot (FDP) on Barro Colorado Island, Panama (Ecological Archives E087-113-S1). Ecology, 87(7) Supplement, 2006, pp. S100–S108 Ó 2006 by the Ecological Society of America

PHYLOGENETIC CLUSTERING AND OVERDISPERSION IN BACTERIAL COMMUNITIES

1,3 2 M. CLAIRE HORNER-DEVINE AND BRENDAN J. M. BOHANNAN 1School of Aquatic and Fishery Science, University of Washington, Seattle, Washington 98195 USA 2Department of Biological Science, Stanford University, Stanford, California 94305 USA

Abstract. Very little is known about the structure of microbial communities, despite their abundance and importance to ecosystem processes. Recent work suggests that bacterial biodiversity might exhibit patterns similar to those of plants and animals. However, relative to our knowledge about the diversity of macro-organisms, we know little about patterns of relatedness in free-living bacterial communities, and relatively few studies have quantitatively examined community structure in a phylogenetic framework. Here we apply phylogenetic tools to bacterial diversity data to determine whether bacterial communities are phylogenetically structured. We find that bacterial communities tend to contain lower taxonomic diversity and are more likely to be phylogenetically clustered than expected by chance. Such phylogenetic clustering may indicate the importance of habitat filtering (where a group of closely related species shares a trait, or suite of traits, that allow them to persist in a given habitat) in the assembly of bacterial communities. Microbial communities are especially accessible for phylogenetic analysis and thus have the potential to figure prominently in the integration of evolutionary biology and community ecology. Key words: bacteria; community structure; microbial ecology; phylogenetic clustering and overdispersion; phylogenetic diversity; phylogenetic structure; relatedness.

INTRODUCTION insight into the relative importance of evolutionary and Although there may be millions of species of bacteria ecological forces in shaping communities (Elton 1946, on Earth, we are only beginning to investigate patterns Webb et al. 2002, Cavender-Bares and Wilczek 2003). in their diversity (Horner-Devine et al. 2004a). Under- The idea that closely related taxa are more likely to standing patterns of bacterial diversity is of particular interact intensely with each other than with more importance, because bacteria likely comprise the major- distantly related taxa is an old one (Darwin 1859); more ity of the planet’s biodiversity, they mediate many recently, this idea has been expanded to suggest that environmental processes that sustain life, and their interspecific interactions are influenced by the net diversity is of great importance in medicine, agriculture, ecological similarity of taxa, and closely related taxa and industry. Recent evidence suggests that bacteria can tend to be more similar ecologically than distantly exhibit patterns in taxonomic diversity and community related taxa (Harvey and Pagel 1991). For example, co- composition similar to those of plants and animals (e.g., occurring rainforest tree species have been observed to Horner-Devine et al. 2003, 2004b). However, most of be more closely related than expected by chance (Webb these studies have relied on measures of diversity that do 2000); such a pattern of phylogenetic attraction or not consider phylogenetic relatedness (Bohannan and clustering can indicate that these closely related taxa Hughes 2003), and few studies have quantitatively share traits important for their persistence in a examined bacterial communities within a phylogenetic particular environment (Webb et al. 2002). Such habitat context (Martin 2002, Davelos et al. 2004, Martin et al. filtering is important and might be more important than 2004). competition, in maintaining rain forest tree species Phylogenetic measures can reveal differences in the diversity (see also Tofts and Silvertown 2000, Webb richness or composition of two communities that would 2000, Kembel and Hubbell 2006). be identical using standard measures of species richness In contrast, a community could be composed of and composition (Martin 2002). Phylogenetic analyses distantly related taxa as a result of current or past of diversity have proven valuable in studies of plant and competitive exclusion between similar (and thus closely animal diversity, because such an approach can lend related) taxa and/or as a result of convergent evolution in traits important for persistence in a given environment (Cavender-Bares et al. 2004, Kembel and Hubbell Manuscript received 9 February 2005; revised 13 July 2005; 2006). However, even for macro-organisms, relatively accepted 12 August 2005; final version received 7 September few studies have quantitatively examined community 2005. Corresponding Editor (ad hoc): J. B. Losos. For reprints of this Special Issue, see footnote 1, p. S1. structure in a phylogenetic framework (but see other 3 E-mail: [email protected] articles this issue), and even fewer have done so for S100 July 2006 RELATEDNESS IN BACTERIAL COMMUNITIES S101 microbes (but see Martin 2002, Francis et al. 2003, previously described sites in northern Virginia (Abbot’s Martin et al. 2004). We know of no other microbial Pitt) and central Delaware (Zhou et al. 2002) at both the studies that employ the approach used here to quantify soil surface (depth ¼ 0.05 m; GenBank accession community structure. numbers AY280351–AY289492) and subsurface (ap- Bacteria offer a unique opportunity to examine the proximately at the depth of the water table, depth . 4.0 phylogenetic structure of multiple communities, because m; GenBank accession numbers AY456755–AY456883 most recently published bacterial diversity data are and AY456885–AY456903) for a total of five samples molecular in nature and thus can be more easily (note that two samples were collected from the subsur- interpreted within a phylogenetic context than data face of Dover Air Force Base; hereafter, subsurface-D1 from many community studies of macro-organisms and subsurface-D2). (Bohannan and Hughes 2003). A large proportion of The third data set consists of 16S rDNA sequences microbes cannot be cultured with current laboratory sampled specifically from ammonia-oxidizing bacteria in techniques (Brock 1987), and thus bacterial taxa are Costa Rican soils (Carney et al. 2004). Samples were often identified from the sequences of indicator genes collected from three land use types on sandy loam soils: extracted from environmental samples (Stackebrandt forest, pasture, and tree plantations. The tree plantation and Goebel 1994). sites included three different site types that differed in Here we take a quantitative approach to examining plant community composition and richness. The one- the phylogenetic structure of bacterial communities from species sites contained only Cordia alliodora, the three- a number of different environments. We ask whether species sites contained C. alliodora, an herb, and a palm, bacterial communities exhibit phylogenetic structure and the five-species sites contained C. alliodora, two (e.g., significant degrees of clustering or overdispersion palm species, and two other hardwoods. Each of the five of taxa across a phylogenic tree) and whether such site types (forest, pasture, and the three plantation patterns vary along environmental gradients. treatments) was replicated three times, with the excep-

METHODS tion of the five-species site, which had two replicates. For each site type, a composite soil sample was collected Data from each of the replicate plots for a total of 14 samples. We used existing bacterial sequence data from four Partial 16S rDNA sequences from these samples were different environments and for three different genes. We deposited in GenBank under accession numbers selected data sets that were of high resolution (e.g., AY631475–AY631851. cloned and sequenced DNA sequences, rather than The fourth data set consists of sequences of functional gradient gel or restriction fragment length data), that genes amplified from five sediments samples collected were from extensively sampled communities (relative to along a salinity and nitrogen gradient in the Chesapeake many studies of bacteria diversity), and that were Bay (Francis et al. 2003; C. A. Francis, J. C. Cornwell, replicated or spanned ecologically interesting environ- and B. B. Ward, unpublished manuscript). One of these mental gradients in aquatic, soil, and sediment habitats. functional genes (amoA) codes for a subunit of ammonia The first data set consists of partial 16S rDNA monooxygenase, an enzyme found only in ammonia- sequences (the most common indicator gene used for oxidizing bacteria (bacteria that mediate the trans- bacterial diversity studies) sampled from freshwater formation of ammonia into nitrite). A 450 base pair mesocosms that span a productivity gradient (Horner- (bp) region was chosen for phylogenetic analysis, Devine et al. 2003). Each mesocosm consisted of a 2 m representing 150 amino acids (GenBank accession diameter polyethylene cattle tank with a screen cover, numbers AY352899–AY353054; Francis et al. 2003). filled with well water. Each mesocosm was inoculated The second functional gene (nirS) codes for a subunit of from the same-pooled sample collected from six ponds nitrite reductase, an enzyme found in denitrifying in southern Michigan that spanned a natural gradient in bacteria (bacteria that mediate the transformation of primary productivity. A gradient of primary productiv- nitrite into nitrogen gas). A 233-bp region was used for ity was established across the mesocosms by maintaining analyses (C. A. Francis, J. C. Cornwell, and B. B. Ward, otherwise identical mesocosms with different input unpublished manuscript). Both gene fragments span the concentrations of nitrogen and phosphorus. At the end active site of their respective proteins (Berks et al. 1995, of a 4-mo growing season, one composite water column Rotthauwe et al. 1997, Braker et al. 2000). sample was used to generate a clone library from each of For each data set, we screened sequences for chimeras the five mesocosms. We selected ;100 clones from each and aligned them using the 2002 version of the ARB of these libraries and sequenced 500 nucleotides from the software package (for 16S genes; available online)4 or 50 terminal of each clone (GenBank accession numbers Sequencher software (for functional genes; Gene Codes DQ064816–DQ065575). Corporation, Ann Arbor, Michigan, USA). We used The second data set consists of 16S rDNA sequences only unambiguously aligned positions to construct the sampled from soil communities at different depths that differed in water saturation and total organic carbon (Zhou et al. 2002). Soil cores were collected from 4 hhttp://www.arb-home.de/i S102 M. C. HORNER-DEVINE AND B. J. M. BOHANNAN Ecology Special Issue phylogenetic hypotheses, and duplicate sequences were K81ufþIþG as the best model of sequence evolution not used when generating phylogenetic trees. Thus for the amoA sequences and TVMþIþG for the nirS sample sizes represent the number of unique sequences sequences. The 16S rDNA trees were constructed using observed, rather than the total number of sequences neighbor-joining distance clustering with a HKY þ analyzed. gamma substitution model (Hasegawa et al. 1985), where gamma was estimated from the data. We used Analysis PAUP* to construct trees for all data sets (Swofford We used indices of community phylogenetic structure 2002). Maximum likelihood methods were used to to compare these communities (Webb 2000). The net estimate branch lengths based on the above HKY and relatedness index (NRI) and nearest taxa index (NTI) gamma DNA substitution models. Trees were boot- measure the degree of phylogenetic clustering of taxa strapped to examine phylogenetic robustness. across a phylogenetic tree in a given sample relative to We also examined how phylogenetic clustering varies the regional pool of taxa. Positive values indicate that a along environmental gradients. NRI and NTI values community is clustered, whereas negative values indicate were standardized by the mean expected value for the that community members are evenly spread or over- number of taxa found in each community (Webb et al. dispersed across a phylogenetic tree. In other words, a 2002). We then used regression and ANOVA imple- positive NRI value indicates a community where mented in JMP, version 4.0, to examine the relationship members are on average more closely related to one between clustering values and environmental parameters another than they are to members of the regional taxon (Sokal and Rohlf 1995). Where data were not normally pool. Such a community thus appears to be clustered on distributed (as determined by the Shapiro-Wilk W test), a phylogenetic tree of the regional taxa. The NRI we used the Kruskal-Wallis one-way analysis of variance measures overall clustering across the phylogeny as the by ranks (Sokal and Rohlf 1995). The following average distance between all pairs of taxa in a environmental parameters were considered: chlorophyll community. Specifically, NRI ¼(Xnet – X(n))/SD(n), a (for the mesocosm data from Horner-Devine et al. where Xnet is the mean phylogenetic distance, measured [2003]), carbon content (for the soil community data as the mean pairwise branch lengths and thus a measure from Zhou et al. [2002]), plant diversity and ammonia of pairwise sequence divergence, between all pairs of n (for the ammonia oxidizer data from Carney et al. taxa in a particular community; X(n) and SD(n) are the [2004]), ammonia and salinity (for the amoA data), and mean and standard deviation of phylogenetic distance nitrate and salinity (for the nirS data). These environ- for n taxa randomly distributed on the phylogeny. We mental parameters were identified in the respective prior obtain these latter values by 1000 random draws from studies as important to taxonomic richness and/or the entire pool of taxa in the phylogeny. Alternatively, community composition. NTI measures the extent of terminal clustering on the phylogeny by determining the minimal distance or RESULTS branch length between taxa in a particular community. Phylogenetic structure The two indices are calculated similarly, except NTI We observed that most of the bacterial communities substitutes Xnear for Xnet, where Xnear is the shortest mean distance between all pairs of n taxa in a we examined exhibited significant phylogenetic structure community sample. We calculated NRI and NTI using (i.e., bacteria tended to co-occur with other bacteria Phylocom 3.22 (Webb et al. 2005). that were more closely related than expected by chance). High and positive values of these indices indicate For example, bacterial communities from freshwater clustering of taxa across the overall phylogeny, whereas mesocosms exhibited significant and positive net relat- low or negative values indicate overdispersion of taxa edness index (NRI) and nearest taxa index (NTI) values across the phylogeny. We tested whether these values (Table 1). This was true when all bacteria were con- (and thus whether the extent of clustering) significantly sidered, as well when the three most common groups of differed from that of a randomly assembled community bacteria sampled from each of the mesocosms (Alpha- with a null model (1000 permutations of randomly proteobacteria, Betaproteobacteria, and Cytophaga- drawn communities). We used a two-tailed significance Bacteroides-Flavobacteria, or CFB) were considered test to evaluate the rank of observed values at P ¼ 0.05, separately. While there was some variation in related- such that an observed rank of ,25 or .975 was ness among the different communities and groups of assumed to be significant overdispersion or clustering, taxa, bacteria in the three most common taxonomic respectively. groups in these communities tended to be clustered. Calculation of NRI and NTI relies on a community Soil communities sampled at different depths showed phylogeny. We used ModelTest 3.06 to determine the different patterns of phylogenetic structure (Table 2). best models of sequence evolution for the unique amoA Subsurface soil communities showed significant clustering and nirS sequences from the Chesapeake Bay sediment for both NRI and NTI. In contrast, one surface sample samples (Posada and Crandall 1998). Using the Akaike was randomly structured phylogenetically, and the other Information Criterion (Akaike 1973), we selected exhibited significant overdispersion for both indices. July 2006 RELATEDNESS IN BACTERIAL COMMUNITIES S103

TABLE 1. Net relatedness index (NRI) and nearest taxa index TABLE 3. The NRI and NTI results for the Costa Rican soil (NTI) results for the 16S rDNA sequences from the five nitrifiers. freshwater mesocosm communities. Group Community N NRI NRI_gt NTI NTI_gt Community N NRI NRI_gt NTI NTI_gt Forest F1 18 0.58 264 0.77 751 All bacteria Forest F2 18 0.05 508 0.77 755 1 108 1.47* 934 5.54* 999 Forest F3 18 0.46 314 1.4 937 2 114 7.28* 999 4.95* 999 Pasture PF 20 2.06* 26 0.16 394 3 87 2.47* 988 2.81* 998 Pasture PR 20 2.42* 9 2.41* 10 4 117 2.18* 13 3.18* 998 Pasture PS 19 4.71* 0 3.76* 0 5 104 3.8* 998 2.51* 998 One One1 29 1.99* 23 1.53 959 One One2 33 2.73* 997 1.84* 986 Betaproteobacteria One One3 34 2.32* 996 1.26 882 1 18 2.34* 991 2.71* 999 Three Three1 36 2.97* 998 1.93* 987 2 35 4.62* 999 0.87 814 Three Three2 35 1.19 887 0.45 332 3 2 1.02 76 1.36 77 Three Three3 31 2.43* 995 1.8* 983 4 19 0.47 690 1.89 972 Five Five1 30 0.91 802 0.3 588 5 7 0.31 627 0.98 825 Five Five2 27 2.5* 998 2.25* 999 Alphaproteobacteria Notes: ‘‘One,’’ ‘‘Three,’’ and ‘‘Five’’ indicate the three 1181.48 74 2.44* 998 plantation treatments containing the respective number of 2 28 2.96* 999 1.97* 994 different species. Community labels describe the group and 3 14 1.53 935 1.35 913 sample number. Other abbreviations and symbols are as in 4332 25 1.94* 994 Table 1. 5 42 4.03* 999 2.35* 998 * Communities that are significantly structured at the P ¼ Cytophaga-Bacteroides-Flavobacteria (CFB) 0.05 level. 1 26 3.44* 999 4.57* 999 2 20 2.52* 996 3.29* 999 tended to be phylogenetically clustered overall with no 3 13 1.21 889 0.48 673 4 24 2.38* 996 2.68* 996 clear pattern in the NTI. 5 7 0.08 504 0.78 234 Finally, sediment bacterial communities sampled at Notes: N ¼ no. taxa in a community. NRI_gt and NTI_gt five sites in the Chesapeake Bay were phylogenetically represent the number of times the observed NRI and NTI structured (i.e., clustered) and contained less genetic values for a community, respectively, were greater than the diversity than a randomly assembled community value for randomly permuted communities. (Table 4). This was true for both the ammonia-oxidizing * Communities that are significantly structured at the P ¼ 0.05 level. bacteria and denitrifying bacteria sampled. All but one Communities that are significantly structured at the P ¼ sample showed significant overall phylogenetic structure 0.10 level. (as estimated by NRI). Interestingly, one community of denitrifying bacteria exhibited significant overdispersion Ammonia-oxidizer communities from Costa Rican as measured by NRI. Phylogenetic clustering measured soils exhibited the most variation in phylogenetic by NTI was more common for denitrifying bacteria than for ammonia-oxidizing bacteria. Only one of the five structure of all the data sets considered (Table 3). While communities from forest soils showed no significant TABLE 4. The NRI and NTI results for the five Chesapeake phylogenetic structure, pasture communities tended to sediment communities. be overdispersed. Communities from the experimental plant treatments with one, three, or five plant species Community N NRI NRI_gt NTI NTI_gt amoA CB1 24 3.58* 999 0.64 275 TABLE 2. The NRI and NTI results for the 16S rDNA CB2 18 5.66* 999 2.79* 999 sequences from soil communities at different depths. CB3 22 6.17* 999 0.47 688 CT1 26 5.04* 999 0.88 789 Community N NRI NRI_gt NTI NTI_gt CT2 14 1.95* 965 1.56 940 Subsurface A 43 3.15* 999 3.72* 999 nirS Subsurface D1 27 2.59* 994 2.97* 999 CB1 79 4.66* 0 0.32 363 Subsurface D2 20 3.81* 999 3.81* 999 CB2 46 5.13* 999 3.56* 998 Surface D 66 3.4* 0 1.88 27 CB3 53 4.27* 999 3.45* 999 Surface A 65 0.98 159 0.29 382 CT1 75 0.39 631 2.45* 990 CT2 87 3.09* 999 5.54* 999 Notes: Labels D and A refer to samples collected at Dover Air Force Base (Delaware, USA) and Abbot’s Pitt (Virginia, Note: Sampling stations were located in the Choptank River USA), respectively. As two subsurface samples were collected at (CT) as well as in the main channel of the Chesapeake Bay Dover Air Force Base, they are denoted D1 and D2. Other (CB). CT1 was located in the upper Choptank, while CT2 was abbreviations and symbols are as in Table 1. located in the lower Choptank. Main channel stations were * Communities that are significantly structured at the P ¼ located in the north bay (CB1), mid-bay (CB2), and south bay 0.05 level. (CB3). Other abbreviations and symbols are as in Table 1. Communities that are significantly structured at the P ¼ * Communities that are significantly structured at the P ¼ 0.10 level. 0.05 level. S104 M. C. HORNER-DEVINE AND B. J. M. BOHANNAN Ecology Special Issue

was a negative trend between NRI estimated for Betaproteobacteria and primary productivity in aquatic mesocosms (Fig. 1A). Relatedness did not vary with productivity for any of the other bacterial groups examined in these mesocosms (results not shown). Phylogenetic structure varied significantly with depth for soil communities (NRI, t ¼ 5.319, df ¼ 3, P ¼ 0.013; NTI, t ¼ 6.693, df ¼ 3, P ¼ 0.0068). In addition, communities sampled from soils with high total organic carbon had lower relatedness values than those sampled from low total organic carbon soils (Fig. 1B). In the Chesapeake Bay, relatedness of denitrifying bacteria exhibited a nonsignificant trend with nitrate and salinity (Fig. 1C for nitrate results; salinity: NRI, NS, results not shown; NTI, R2 ¼ 0.5121, P ¼ 0.107). In contrast, relatedness measures of ammonia-oxidizing bacteria did not vary with any environmental parameters measured (ammonium and salinity, results not shown). Phylogenetic structure of communities of ammonia- oxidizing bacteria varied with plant community composition in Costa Rican soils for NRI (ANOVA, F4,9 ¼ 5.507, MSE ¼ 2.33, P ¼ 0.0160), but not for NTI (Kruskal-Wallis: v2 ¼ 6.5095, df ¼ 4, P ¼ 0.164). Pairwise post-hoc comparisons of the different treatments revealed that the bacterial communities from sites with one, three, or five focal plant species were more clustered than pasture communities as measured by NRI. There was a weak positive relationship between ammonia and NTI, but not NRI, for these bacteria (R2 ¼ 0.218, P ¼ 0.052).

DISCUSSION Our results suggest that bacteria tend to co-occur with other closely related bacteria more often than expected by chance, as has been observed for some plant species (Webb 2000; also see Cavender-Bares et al. 2006, Kembel and Hubbell 2006, Lovette and Hochachka 2006, Weiblen et al. 2006). In addition, we observed that phylogenetic structure can vary along environmental gradients. FIG. 1. Variation of relatedness along environmental We observed significant net relatedness index (NRI) gradients. (A) Relatedness decreased with increasing productivity in freshwater mesocosms for Betaproteobacteria (NRI, R2 and nearest taxa index (NTI) values for freshwater ¼ 0.829, P ¼ 0.0588; mesocosm three was excluded due to small bacterial communities from experimental mesocosms. community size). (B) Relatedness decreased with total organic Relatedness information provides a different window 2 carbon in bacterial soil communities (NRI, R ¼ 0.925, P ¼ into bacterial communities than does information 0.0058; NTI, R2 ¼ 0.966, P ¼ 0.0017). (C) There was a significant negative relationship between the relatedness of nirS concerning richness or taxonomic composition. Accord- genes and nitrate in Chesapeake Bay sediment communities ingly, our observations that the relatedness of co- (NRI, R2 ¼ 0.909, P ¼ 0.0077; NTI, R2 ¼ 0.601, P ¼ 0.0768). occurring Betaproteobacteria decreases with productivity, is in contrast to previous observations that their taxonomic richness does not (Horner-Devine et al. ammonia-oxidizing communities sampled showed sig- 2003). We observed that each of the five communities nificant phylogenetic clustering as estimated by NTI. contained approximately the same number of taxa regardless of productivity, but these taxa tended to be Phylogenetic structure and the environment more distantly related at higher productivities. Decreas- ing relatedness with increasing productivity might We also observed that measures of phylogenetic indicate that low productivity environments are more structure can vary along environmental gradients. There ‘‘stressful’’ (e.g., impose a stronger ‘‘filter’’ on a July 2006 RELATEDNESS IN BACTERIAL COMMUNITIES S105 community) for Betaproteobacteria than do more Phylogenetic scale (the taxonomic group or rank productive environments. In contrast, we did not under consideration) has been shown to influence the observe a relationship between relatedness of Alpha- observation of phylogenetic patterns (Silvertown et al. proteobacteria or Cytophaga-Bacteroides-Flavobacteria 2001, 2006, Cavender-Bares et al. 2004, 2006). However, (CFB) and productivity in the mesocosm study, despite the effect of phylogenetic scale was not evident for our previous observations of changes in taxonomic bacteria from the freshwater mesocosms. We tended to richness of these groups with productivity (Horner- observe phylogenetic clustering both when we examined Devine et al. 2003). all bacteria together and when we examined taxonomic Clustered distributions have been interpreted as subsets of the bacteria (Alphaproteobacteria, Betapro- evidence of habitat filtering, where a group of closely teobacteria, and CBF). It is possible that Alphaproteo- related species shares a trait, or suite of traits, that allow bacteria, Betaproteobacteria, and CBF encompass such them to persist in a given habitat (Webb et al. 2002). a broad range of bacterial ecotypes that, even with the Alternatively, significant phylogenetic clustering could NTI (which focuses on terminal clustering and thus is be the result of differential dispersal and/or colonization particularly sensitive), it is less likely that one will abilities, or an adaptive radiation event. While it is observe overdispersion. beyond the scope of this work to determine the process Recent work by Zhou et al. (2002) and Treves et al. responsible for the clustering we observed, the results (2003) suggests that spatial isolation plays an important from the freshwater mesocosms suggest that habitat role in structuring soil bacterial communities. They filtering, rather than an adaptive radiation event or observed that unsaturated, surface communities had colonization effects, was important in the assembly of more uniform rank abundance patterns than did these bacterial communities. In this mesocosm study, communities from saturated, subsurface communities, bacterial communities were established in each meso- which exhibited high-dominance distributions (Zhou et cosm from the same inoculum of bacteria from natural al. 2002). They interpreted the uniform distribution pond communities (Horner-Devine et al. 2003). Thus, from the surface samples as evidence that local the history of colonization could not play a role in the competition does not play a significant role in structur- patterns we observed. Although dispersal among meso- ing the soil communities they studied. Their observa- cosms was not prevented (and likely occurred), the tions (and subsequent mathematical modeling and productivity gradient was randomized in space (i.e., laboratory experimentation) suggested that spatial iso- mesocosms with similar productivities were not clustered lation might limit competition in the surface soils in space), and thus clinal dispersal is unlikely to underlie (Treves et al. 2003). Our results do not support this the patterns we observed. Finally, since the mesocosm hypothesis. We observed phylogenetic clustering in the communities were sampled four months after they were subsurface samples, where spatial isolation was pre- initiated, it is unlikely that the ribosomal gene evolved dicted to be minimized due to high water content and during the course of the experiment, and thus it is thus where there was an expectation for strong unlikely that the clustering is due to adaptive radiation competition and phylogenetic overdispersion. In con- during the experiment. Without such information for the trast we observed phylogenetic overdispersion in one of other data sets we analyzed, it is difficult to distinguish the surface samples, where isolation was predicted to be among habitat filtering, adaptive radiation, or coloni- high and competition weak. zation processes in the systems that were not manipu- We did observe that phylogenetic clustering decreased lated. However, the results from the mesocosm study with increasing total organic carbon (which covaried suggest that habitat filtering could be an important force with depth) in the Zhou et al. (2002) soil data set. Thus in the assembly of at least some bacterial communities. as carbon availability increased, the strength of cluster- Overdispersion of taxa across a phylogeny has been ing and perhaps habitat filtering decreased. The observed in natural communities (Ackerly et al. 2006, potential decrease in the strength of filtering is unlikely Cavender-Bares et al. 2006, Silvertown et al. 2006) and to be related to an increase in the role of competition for could indicate that negative interactions (e.g., competi- carbon, since competition would likely decrease with tion) are important in community assembly (Graves and increasing carbon. More information on the types of Gotelli 1993, Webb et al. 2002). Although we did carbon present, as well as C:N ratios, might lend more observe significant phylogenetic overdispersion in one of insight into the underlying processes. the freshwater bacterial communities, our observations We observed both phylogenetic clustering and over- do not suggest that competition played an overwhelming dispersion for ammonia-oxidizing bacteria from Costa role in structuring the communities we studied at the Rican soils. It is possible that, for ammonia-oxidizing scales examined here. However, it is important to note bacteria, a more restricted (and potentially more that habitat filtering and competition likely act in ecologically similar) group of taxa, it might be easier concert to produce the communities we observe. Thus to detect interactions among taxa, because ammonia- even where the phylogenetic signature suggests the oxidizing bacteria likely compete for similar resources. importance of habitat filtering, local competition can Carney et al. (2004) found that neither bacterial richness also be occurring. nor composition changed across plant diversity treat- S106 M. C. HORNER-DEVINE AND B. J. M. BOHANNAN Ecology Special Issue ments (one, three, or five focal plant species). Similarly, Morgan et al. 2001, Prinzing et al. 2001), but not all we did not observe pairwise differences in phylogenetic (Losos et al. 2003, Rice et al. 2003, Knouft et al. 2006). structure among the plant treatments using post-hoc How universally the assumption about similarity of comparisons. Carney et al. (2004) also observed differ- closely related organisms applies to microorganisms is ences in the ammonia-oxidizer community among land- currently unknown. If this assumption does not hold for use types in some measures of diversity and in most bacteria, other explanations might be necessary for composition. We observed that, while pasture and forest the patterns we observe. For example, some bacteria are did not differ in phylogenetic clustering, pasture capable of lateral gene transfer (LGT; Ochman et al. communities were less phylogenetically diverse (i.e., less 2000, Lerat et al. 2003). Lateral gene transfer among co- clustered) than each of the plant treatments. In fact, occurring bacteria could weaken or uncouple the pasture communities tended to be overdispersed. We relationship between ecological similarity and evolu- also found a weak positive relationship between terminal tionary relatedness, if ecologically relevant genes are clustering and ammonia, such that clustering (and exchanged more often than phylogenetically informative perhaps the importance of habitat filtering rather than housekeeping genes (e.g., ribosomal genes) as has been competition) increased with ammonia. This is consistent suggested (Lerat et al. 2003). Rampant LGT would with an increase in the importance of habitat filtering reduce the prevalence of phylogenetic clustering or (and conversely, perhaps a decrease in the relative overdispersion due to ecological processes. However, strength of competition) for ammonia as ammonia we observed a significant level of phylogenetic clustering concentrations increased. in the communities that we examined, suggesting that Analysis of amoA and nirS genes sampled from the LGT does not substantially overwhelm phylogenetic Chesapeake Bay offered us an opportunity to examine patterns in these communities. In addition, recent work community patterns deduced from potentially ‘‘ecolog- in environmental genomics suggests that on recent ically relevant’’ genes. We assumed that phylogenetic evolutionary time scales horizontal gene transfer is not overdispersion would be more prevalent in such data, rampant in natural microbial communities (Lerat et al. where the taxa sampled are essentially from a single 2003). guild (i.e., a group performing the same function and Martin et al. (2004) used a different approach requiring the same resources). However, we did not (lineage-per-time analysis) to look for phylogenetic observe overdispersion for amoA, and we observed patterns in microbial diversity data. They failed to show overdispersion in only one sample of nirS. In hindsight significant phylogenetic structure (i.e., an overabun- this is not overly surprising for nirS, given that dance of closely related or distantly related sequences) denitrifying bacteria can be ecologically very different across several different data sets. However their study despite sharing the nirS gene (Shapleigh 2000). However, differed from ours in that they assumed a ‘‘universal’’ ammonia-oxidizing bacteria are believed to be a null model (an exponential increase in lineages) for all physiologically constrained group, existing solely on data sets, rather than creating a null expectation for each the oxidation of ammonia. The lack of overdispersion data set by resampling of a regional phylogenetic tree. In suggests that the amoA gene may be too conserved to the approach used here, we are interested in whether reveal ecological differences or that sequence variation observed communities differ in phylogenetic diversity at this scale does not reflect ecological differences. from communities created by a random draw from the Previous analysis of the amoA samples demonstrated available taxa in the regional pool. The Martin et al. a nonsignificant trend toward decreasing richness with (2004) approach suffers from a lack of power if the increasing salinity (Francis et al. 2003). We did not fraction of diversity sampled is small, making the task of observe a relationship between phylogenetic structure as detecting phylogenetic structure very difficult. While the inferred from this gene and salinity or ammonia. In communities examined here are also undersampled, the contrast, we observed a significant decrease in phyloge- use of a relative measure of community relatedness netic clustering as inferred from the nirS gene with decreases the influence of undersampling, provided increasing nitrate and a weak relationship between communities in a given analysis are sampled with equal clustering and salinity. It is difficult to interpret the effort. Furthermore, if the questions of interest concern relationship with nitrate; one might expect that increas- community assembly (as they do in our study), assuming ing nitrate availability would lead to decreased com- a null model based on regional tree resampling is petition for this substrate by denitrifiers or stronger appropriate because it should model the assembly habitat filtering for denitrifying bacteria with genes that process. confer an advantage at high nitrate concentrations; we We have observed that bacterial communities exhibit observed the opposite trend. phylogenetic structure, in some cases similar to that The interpretations of our results are based on the observed for plants, and that this structure can vary assumption that closely related taxa are more ecologi- along environmental gradients. Our results suggest that cally similar than distantly related taxa. This assumption habitat filtering might be relatively more important to has been shown to be true for some plants, animals, and the assembly of bacterial communities than competition. microbes (Kuittinen et al. 1997, Nubel et al. 1999, Why might this be the case? Recent work suggests that July 2006 RELATEDNESS IN BACTERIAL COMMUNITIES S107 the greater the degree of environmental heterogeneity phylogenetic structure for microbial and macrobial over which one samples a community, the more likely communities, it is possible that similar mechanisms that phylogenetic clustering, rather than overdispersion, could be responsible for the structure of communities will be present (Cavender-Bares et al. 2006, Silvertown from these very different forms of life. et al. 2006). The data sets used in our study consisted of ACKNOWLEDGMENTS samples that were extremely large relative to the size of the target organisms and the scales over which We are grateful to David Ackerly, Will Cornwell, Clara Davis, and Cam Webb for many informative discussions about individuals interact (as is the case in most studies of phylogenetic structure and community assembly. We would microbial diversity) and thus likely included substantial also like to thank Karen Carney, Chris Francis, and Jizhong environmental heterogeneity. As such, the spatial scale Zhou for allowing us to use their data for these analyses. of sampling might bias the results towards clustering Finally, we thank Jonathan Losos and two anonymous rather than overdispersion. reviewers for their comments and suggestions. M. C. Horner- Devine was supported by funding from the American Associ- Recent studies have also suggested that phylogenetic ation of University Women and the Center for Evolutionary scale should affect the prevalence of clustering; increas- Studies at Stanford. This work was also supported by a grant ing phylogenetic scale (i.e., an increase in taxonomic from the NSF (DEB0108556) to B. Bohannan. lumping) might result in an increased prevalence of LITERATURE CITED clustering (Silvertown et al. 2006). Substantial ecological Ackerly, D. D., D. W. Schwilk, and C. O. Webb. 2006. Niche diversity has been shown to be present within microbial evolution and adaptive radiation: testing the order of trait taxa defined using molecular markers, especially riboso- divergence. Ecology 87:S50–S61. mal markers (e.g., Ward et al. 1998, Rocap et al. 2002). Akaike, H. 1973. Information theory and an extension of the The molecular approach used to characterize microbial maximum likelihood principle. 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PHYLOGENETIC STRUCTURE OF FLORIDIAN PLANT COMMUNITIES DEPENDS ON TAXONOMIC AND SPATIAL SCALE

1 JEANNINE CAVENDER-BARES, ADRIENNE KEEN, AND BRIANNA MILES Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, Minnesota 55108 USA

Abstract. Consideration of the scale at which communities are defined both taxonomically and spatially can reconcile apparently contradictory results on the extent to which plants show phylogenetic niche conservatism. In plant communities in north central Florida, we collected species abundances in 55 0.1-ha plots in several state parks. When communities were defined narrowly to include a single phylogenetic lineage, such as Quercus, Pinus,orIlex, neighbors tended to be less related than expected (phylogenetic overdispersion) or there was no pattern. If the same communities were defined more broadly, such as when all seed plants were included, neighbors tended to be more related than expected (phylogenetic clustering). These results provide evidence that species interactions among close relatives influence community structure, but they also show that niche conservatism is increasingly evident as communities are defined to include greater phylogenetic diversity. We also found that, as the spatial scale is increased to encompass greater environmental heterogeneity, niche conservatism emerges as the dominant pattern. We then examined patterns of trait evolution in relation to trait similarity within communities for 11 functional traits for a single phylogenetic lineage (Quercus) and for all woody plants. Among the oaks, convergent evolution of traits important for environmental filtering contributes to the observed pattern of phylogenetic overdispersion. At the broader taxonomic scale, traits tend to be conserved, giving rise to phylogenetic clustering. The shift from overdispersion to clustering can be explained by the increasing conservatism of traits at broader phylogenetic scales. Key words: environmental heterogeneity; Florida; Ilex; niche conservatism; overdispersion; phylogenetic structure of communities; Pinus; Quercus; taxonomic scale; trait convergence.

INTRODUCTION 1946, MacArthur and Levins 1967, Chesson 1991, There is growing recognition that species evolve Leibold 1998). The two processes lead to opposite within communities and that community interactions predictions about the phenotypic similarity and phylo- influence the evolutionary process (Antonovics 1992, genetic relatedness of co-occurring species (Tofts and Neuhauser et al. 2003, Whitham et al. 2003). At the Silvertown 2000, Webb et al. 2002). If closely related same time, we are becoming increasingly aware that species share similar physiological limitations and evolutionary processes, particularly the way that traits exhibit evolutionary niche conservatism, environmental filtering will tend to cause closely related species to co- evolve within lineages, influence species distributions occur (phylogenetic clustering). In contrast, competitive and assembly in communities (McPeek 1996, Webb et al. exclusion should limit the coexistence of closely related 2002, Ackerly 2003, Chazdon et al. 2003, Cavender- species if species compete for the same limiting Bares et al. 2004a). This study seeks to understand the resources, leading to the opposite pattern of phyloge- role that trait evolution plays in determining the netic overdispersion. Both processes can operate simul- phylogenetic structure of communities and the extent taneously in real communities, but have greater to which phylogenetic structure depends on how influence at different scales. Keddy and Weiher (1999) communities are defined. hypothesized that limiting similarity should have greater Two processes are often considered as central to the importance at smaller spatial scales, whereas environ- assembly of communities: (1) filtering of species that can mental filtering should predominate at larger spatial persist within a community on the basis of their scales. Evidence supporting this view has been found in tolerance of the abiotic environment (e.g., Weiher and meadow communities in Great Britain (Silvertown et al. Keddy 1995), and (2) competitive interactions among 2005). species that limit their long-term coexistence (Elton At the same time, the ecological process that appears to predominate might also depend on how broadly or Manuscript received 26 January 2005; revised 17 August narrowly communities are defined. For example, phylo- 2005; accepted 18 August 2005. Corresponding Editor: A. A. genetic clustering was found among tree species in Agrawal. For reprints of this Special Issue, see footnote 1, p. S1. rainforest communities in Borneo (Webb 2000), as well 1 E-mail: [email protected] as in herbaceous communities in Great Britain (Tofts S109 S110 JEANNINE CAVENDER-BARES ET AL. Ecology Special Issue

FIG. 1. (A) Phylogenetic structure of communities. An observed correlation (solid line) that is more negative than expected (dashed line) indicates phylogenetic clustering (left panel), because closely related species occur together more often than expected by chance; an observed correlation that is more positive than expected indicates phylogenetic overdispersion (right panel), because closely related species do not occur together. (B) Trait similarity in communities. An observed correlation that is more negative than expected indicates that co-occurring species show similar trait values (phenotypic clustering, left panel); an observed correlation that is more positive than expected indicates that trait values can be highly variable within communities (phenotypic overdispersion, right panel). and Silvertown 2000). Both studies considered a large and environmental filtering. Narrowly defined commun- number of angiosperm lineages, providing support for ities, in contrast, are more likely to show phylogenetic the generalization that evolutionary stasis, or phyloge- overdispersion, either as a result of trait convergence, netic niche conservatism, is widespread among plant trait overdispersion, or both. communities around the globe (Harvey and Pagel 1991, 2) The phylogenetic structure of communities depends Eldridge 1995, Wen 1999, Webb et al. 2002, Ackerly on the spatial scale of the analysis. As the spatial scale is 2003, Qian and Ricklefs 2004). In contrast, phylogenetic increased to encompass greater environmental hetero- overdispersion has been found in narrowly defined geneity, species interactions should become less impor- communities that include a single phylogenetic lineage, tant, and phylogenetic clustering should emerge as the including within Caribbean lizard communities (Losos et dominant pattern. al. 2003) and among co-occurring oaks in north central 3) The relationship between trait evolution (conserva- Florida (Cavender-Bares et al. 2004a). In the latter tive vs. convergent) and trait similarity within commun- study, evidence of convergence, rather than conserva- ities (clustered vs. overdispersed) should predict the tism, in the evolution of species niches highlighted the phylogenetic structure of communities at different scales prevalence of species interactions over niche conserva- of analysis. tism in community assembly. These studies suggest that patterns of phylogenetic dispersion might vary system- MATERIALS AND METHODS atically with taxonomic scale. The present study seeks to Tests of phylogenetic structure of communities evaluate this possibility by examining the vegetation of Florida at various taxonomic and spatial scales. In We examined the phylogenetic structure of commun- addition, the study addresses the role that trait evolution ities by comparing the degree of co-occurrence of species plays in the phylogenetic structure of communities. pairs in relation to the phylogenetic distance between Specifically, we make the following predictions: them (Fig. 1A). Due to nonindependence and non- 1) The pattern of phylogenetic structure among normality of the data points, these correlations were communities depends on how a community is defined compared to null models. Tests for phylogenetic in terms of the taxa included. The more broadly a clustering and overdispersion were conducted for a suite community is defined, the more likely it will be to show of different data sets in north central Florida and in the phylogenetic clustering as a result of trait conservatism entire state of Florida that vary in spatial extent and July 2006 SCALE AND FLORIDIAN PLANT COMMUNITIES S111 resolution of data. We compared the correlation computed from each of the 999 randomized trees, as well coefficient of the relationship between co-occurrence as from the original phylogeny, using a program in and phylogenetic distance of species pairs to a null Visual Basic (J. Cavender-Bares, unpublished software). model in which either species distributions were We used the result of this computation to calculate a permuted or phylogenetic relationships among species null distribution of correlation coefficients between were randomized (Fig. 1A; Cavender-Bares and Wilczek pairwise species co-occurrence and phylogenetic dis- 2003). The pairwise values of co-occurrence (C) were tance. In all analyses, a P value was calculated based on calculated based on proportional similarity (Schoener a two-tailed test. Since the null models use 999 1970) as follows: Cih ¼ 1 – 0.5 Rjpij – phjj, where Cih is the randomizations plus the observed value, the minimum co-occurrence of species i and h, and pij is the proportion P value is 0.002 (Manly 1991). of total basal area or the proportion of occurrences of the ith species in the jth plot. We calculated phylogenetic Trait similarity within communities distances from the estimated intervening branch length We used a similar test to examine trait similarity distances (measured in millions of years) between species within communities (Fig. 1B) by comparing the absolute pairs based on community phylogenies created for each value of pairwise differences in trait values to the degree test (see Phylogenetic analyses). of co-occurrence between species pairs. The observed Three null models were used. The first two generated correlation coefficients were tested against null model 2, a null distribution of expected species occurrence in which the presence/absence of species within plots was patterns against which to compare the observed data. randomized keeping row and column totals constant. These allow us to ask the following question: Given the The ranking of the observed correlation coefficient evolutionary history of the taxa in the regional species relative to that of the null model provides a way to pool, does phylogenetic relatedness influence the way order the measured traits in terms of their similarity that species have assembled in communities? In null within communities. The term ‘‘phenotypic clustering’’ model 1, basal area of species within plots was refers to high trait similarity within communities, while randomized by reshuffling raw data values 999 times the term ‘‘phenotypic overdispersion’’ refers to low trait across plots, but constraining the total basal area per similarity within communities (Fig. 1B) species. Null model 2 used presence/absence data, instead of basal area, and constrained both total oc- The spatial extent of communities currences per species and total number of species per In order to test the importance of (1) the spatial plot, using the sequential swap algorithm (Gotelli and resolution of data, and (2) how communities are defined, Entsminger 2001b). The null model for the presence/ we analyzed a series of community data sets that differ absence data may be the most biologically realistic, in the resolution of species abundances and in the because it constrains both species abundances and plot community definition. First, we examined the influence diversity levels (Gotelli and Graves 1996). However, of using basal area or presence/absence to evaluate presence/absence data provide a lower degree of species abundance in random 0.1-ha plots of north resolution for the distributions of species than basal central Florida. Second, we examined the influence of area, because differences in relative abundance within the way community boundaries are defined by using plots are not considered. A null model that constrains these random 0.1-ha plots, where community bounda- total basal area per species and plot would probably be ries are fixed by a standard area (hereafter referred to as impossible to construct and was not attempted. the plot survey), or by using previously established Randomizations were carried out using Ecosim (Gotelli community classifications and vegetation maps within and Entsminger 2001a), and the co-occurrence vs. state parks (hereafter referred to as community classi- phylogenetic distance correlations were carried out in fications). In the latter case, community boundaries in a self-written Visual Basic program, modified to north central Florida were defined by environmental accommodate large data sets from Cavender-Bares et variation and the vegetation itself according to the al. (2004a). Florida Natural Areas Inventory and the Florida A third null model kept constant the distribution of Department of Natural Resources (FNAI and FDNR species within communities, but randomized the phylo- 1990). Previously determined community classifications genetic tree topology. Null model 3 allows us to ask, for the entire state of Florida were also used, so that given the distribution of species in communities, does environmental variation among communities encom- the phylogenetic relatedness of species within those passes topographic and edaphic variation, as well as communities differ from random expectation? To climatic variation. randomize, we used the ‘‘random branch moves’’ To examine the influence of the inclusion or exclusion algorithm in Mesquite (Maddison and Maddison 2000) of communities on phylogenetic structure, we performed and assigned the number of branch moves to equal the tests of co-occurrence vs. phylogenetic distance for three number of taxa in the tree. The total branch length individual genera (Quercus, Pinus,andIlex), using distance from the basal node to the tips was kept community classification data sets for six state parks in constant. A distance matrix was then automatically north central Florida relative to two null models (both S112 JEANNINE CAVENDER-BARES ET AL. Ecology Special Issue corresponding to null model 2). We created the first null those used in the present study (Ackerly 2000). Never- model by permuting the presence of species across only theless, examination of patterns of trait evolution with those communities that contained a member of the increasing taxonomic diversity is useful for under- genus (no community had zero as a matrix element). We standing how trait conservatism shifts with phyloge- created the second null model by permuting species’ netic scale in a single study system. presences across all communities that existed in the six parks examined, regardless of whether any members of Trait data collection the genus were found in them or not (a number of We measured a suite of leaf traits on mature trees communities were represented by zeros in the matrix). across the range of their local environmental distribu- This allowed us to test whether increasing the number of tions, collected from five state and city parks in north communities (effectively increasing the spatial scale and central Florida, including San Felasco Hammock State the degree of environmental heterogeneity) would alter Preserve, Ichetucknee Springs State Park, Morning Side the results. Nature Center, Payne’s Prairie State Preserve, and O’Leno State Park. For five individuals of each species, Inclusiveness of taxa within communities three sun and three shade leaves from each individual To examine the influence of phylogenetic scale, or the were measured for leaf area, scanned, and dried for leaf inclusiveness of taxa, on phylogenetic structure of mass. We then used scanned leaf images to calculate communities, we carried out the same tests for the plot perimeter (P), the perimeter-to-area ratio (P/A), which survey data to examine the phylogenetic structure of has been shown to be correlated with leaf hydraulic plant species within communities for (1) species within a conductance (Sack et al. 2003), and lobedness (PL/A), monophyletic lineage (Quercus), (2) all angiosperms in determined as the perimeter-to-area ratio, scaled by leaf the same guild (tree and shrub strata), (3) all plants in length (L). In addition, we took plant height, seed mass, the tree and shrub strata, and (4) all recorded plants in and cotyledon type (photosynthetic or storage cotyle- all strata, including trees, shrubs, vines, epiphytes, and dons), which has been shown to influence growth herbaceous species. These were tested against null dynamics (Kitajima and Fenner 2000), from published models 1, 2, and 3, and against null models 2 and 3 (Kurz and Godfrey 1962, Mirov 1967, Schopmeyer only in the latter case, as only presence/absence data 1974, Godfrey and Wooten 1981, Wenger 1983, Godfrey were available. We ran the same tests for the community 1988) or online sources. We were able to collect leaf trait classification data. The three most diverse woody plant data for 90 of the 122 species examined in the plot genera within north central Florida, including the oaks survey; maximum height and leaf habit were obtained (18 species), the hollies (seven species), and the pines (six for 115 species, seed mass for 63 species, and cotyledon species), were included to examine lineage-specific type for 87 species. patterns in the context of their biogeographic and It is useful to include as many different kinds of evolutionary history. traits as possible that might contribute to ecological filtering and interactions among species. The present Trait similarity within communities study, however, examined a limited number of easily in relation to trait evolution measured traits. A suite of additional traits, including For suites of life history and functional traits, we maximum hydraulic conductivity, vessel diameter, used the relationship between trait conservatism and wood density, leaf longevity, and others were available trait similarity within communities to understand the from previous studies for the Quercus species (Cav- phylogenetic structures of communities for the oaks ender-Bares and Holbrook 2001, Cavender-Bares et al. only, as well as for all trees and shrubs in the plot 2004b), and these are presented in the oak analysis for survey. Trait similarity within communities was deter- comparison. mined from the correlation of trait differences between species pairs and their degree of co-occurrence, relative Community data collection to null model 1 (Fig. 1B). Trait conservatism (or phy- Plot survey data in north central Florida.—Quantita- logenetic signal) was calculated using methods devel- tive vegetation data were collected from randomly oped by Ackerly in the ‘‘Analysis of Traits’’ module in located 20 3 50 m (0.1 ha) plots established in a Phylocom (Webb et al. 2004), based on trait differences previous study (Cavender-Bares et al. 2004a, b)in between nodes, normalized by the standard deviation of several state parks in north central Florida. Seventy the trait within a given lineage relative to a null model four plots were originally established in 1998 for a in which species are randomized across the phylogeny study on oaks, and 55 of these original plots were (Moles et al. 2005). We used the ranking of the resampled to determine the basal area of all woody observed phylogenetic signal relative to the simulations species as well as the presence/absence of all plant of the null model to order the traits by their degree of species common enough within a plot to be conspic- conservatism. We recognize that measures of trait uous. Insufficient time and inability to relocate some of conservatism are influenced by taxon sampling and the plots prevented a complete resampling of all of the might be biased in highly pruned phylogenies, such as original plots. Within each plot, the diameter at breast July 2006 SCALE AND FLORIDIAN PLANT COMMUNITIES S113 height of each tree over 1 m height was measured for ities in the analyses, including wetlands and coastal calculation of basal area for a total of 122 tree and communities. Unlike the analyses for the parks in north shrub species (see Appendix A). The presence/absence central Florida, each community was represented only of all seed plants was also recorded for each of the once, regardless of how commonly it occurs. plots (141 species; see Appendix A). The majority of the plots are located in three state parks, including San Phylogenetic analyses Felasco Hammock State Preserve (28 plots), a 2803-ha Phylogenetic reconstruction.—Community phyloge- park in Alachua County; Ichetucknee Spring State nies were created for each test using Phylomatic (Webb Park (13 plots), a 921-ha park bridging Columbia and and Donoghue 2005). Phylomatic is an online applica- Suwanee counties and bisected by the Ichetucknee tion for creating a backbone phylogeny based on family River; and Manatee Springs State Park (12 plots), a and genera.2 The maximally resolved seed plant tree 960-ha park abutting the Suwanee River in Levy used for our trees relies on the online resource County. Two more plots were resampled at other sites continually updated by P. F. Stevens, i.e., Angiosperm in the region, including one at Morning Side Nature Phylogeny.3 Sources for tree construction down to the Center and one at Paynes Prairie Preserve State Park. family level are extensively documented on this web site, An effort was made to sample across a range of the although phylogenies created through the Phylomatic major woody plant community types in north central maximally resolved seed plant tree are not entirely Florida. resolved to the family level. We used a supertree of the Community classification data in north central Flori- angiosperms (Davies et al. 2004) to manually resolve all da.—We obtained species lists for each of six state parks trees to the family level. Higher taxa were resolved in north central Florida and corresponding maps of according to published phylogenies (see Appendix B). If natural community types from the District 2 Park no published information was available to resolve Service in Gainesville, Florida, USA. Parks included polytomies, they remained unresolved. (See Appendix San Felasco Hammock, Ichetucknee Springs, O’Leno C for all reconstructed phylogenies.) It is important to State Park, Big Shoals State Park, Stephen Foster State recognize that the community phylogenies generated Park, and Goldhead Branch State Park. In Florida, 63 using this approach represent only approximations of terrestrial natural communities were identified and true species relationships and should be refined as more defined by the Florida Natural Areas Inventory and data and other methods for constructing supertrees Florida Department of Natural Resources (FNAI and become available. The Quercus phylogeny was based FDNR 1990), 29 of which occur in terrestrial areas of on Cavender-Bares et al. (2004a) and was consistent these state parks in north central Florida. Aquatic with Manos et al. (1999). The four most parsimonious communities, which comprised nearly half of the trees from that analysis were tested, as well as several defined communities, were excluded from the analyses less resolved phylogenies that collapsed nodes with low because plant species were either poorly represented or bootstrap support. We converted branch lengths to poorly documented. A natural community is defined by millions of years. A less resolved topology was used in the FNAI and FDNR (1990) as a ‘‘distinct and the analyses presented here (see Appendix C), but the reoccurring assemblage of populations of plants, results were indistinguishable from those using the animals, fungi, and microbes naturally associated with more resolved phylogenies (data not shown). We based each other and their physical environment.’’ In each the Ilex phylogeny on Cenoud (2000), and the Pinus park, we assigned species to designated communities, phylogeny on Millar (1993), Schwilk and Ackerly either by data directly from the Park Service or from a (2001), Grotkopp et al. (2004), and Gernandt et al. master list from the FNAI and FDNR (1990). If (2005). community assignments were not available from these Branch length estimation.—Branch lengths were based sources, we determined them from two other sources on minimum ages of nodes determined for genera, (Kurz and Godfrey 1962, Godfrey 1988). Data were families, and higher orders from fossil data, and we sufficient to include only the tree and shrub species (216 extrapolated to higher order branches by spacing species). There were 29 possible community types, undated nodes in the tree evenly between dated nodes. replicated according to whether they occurred in multi- This was done using an averaging algorithm in ple state parks, giving a total of 75 sample communities Phylocom (Webb et al. 2004) called ‘‘BLADJ’’ (Branch used for the community classification analysis. Length ADJustment, available online).4 Most of the Community classification data for the state of Flori- node ages at the family level were taken from Wikstro¨m da.—A master species list with the community affili- et al. (2001). At the genus level, we used additional ations of these species was developed for the entire state sources, including Daghlian and Crepet (1983) for of Florida from the FNAI and FDNR report. We ran Quercus, based on the first appearance of the genus in two analyses: one for all listed plant species with identified community types (383 species), and a second 2 hhttp://www.phylodiversity.net/phylomatici for plants in the tree and shrub guild only (255 species) 3 hhttp://www.mobot.org/MOBOT/research/APwebi (see Appendix A). We included 50 terrestrial commun- 4 hhttp://www.phylodiversity.net/bladji S114 JEANNINE CAVENDER-BARES ET AL. Ecology Special Issue

TABLE 1. Tests for patterns of phylogenetic structure for community data sets that vary in how communities are deﬁned. For each data set, the number of taxa, the number of communities, the data type (basal area or presence/absence), and the null model used in the analysis are given.

r Data type and taxa included Communities No. taxa No. communities Data type Null model (observed) North central Florida Plot survey data (three parks, 55 plots) Quercus species all 17 55 basal area 1 0.184 Quercus species all 17 55 pres/abs 2 0.157 Quercus species all 17 55 basal area 3 0.184 Quercus species all 17 55 pres/abs 3 0.157 Angiosperm trees and shrubs all 113 55 basal area 1 0.030 Angiosperm trees and shrubs/no oaks all 96 55 basal area 1 0.019 All tree and shrub species all 122 55 basal area 1 0.036 All tree and shrub secies all 122 55 pres/abs 2 0.043 All tree and shrub species all 122 55 basal area 3 0.036 All tree and shrub species all 122 55 pres/abs 3 0.043 All angiosperms all 130 55 pres/abs 2 0.008 All angiosperms/no oaks all 113 55 pres/abs 2 0.014 All plant species all 141 55 pres/abs 2 0.027 All plant species all 141 55 pres/abs 3 0.027 North central Florida Community classification data (six parks, 29 terrestrial community types) Quercus species all 18 75 pres/abs 2 0.153 Quercus species oak only 18 38 pres/abs 2 0.153 Quercus species all 18 75 pres/abs 3 0.153 Pinus species all 6 75 pres/abs 2 0.284 Pinus species pine only 6 29 pres/abs 2 0.284 Pinus species all 6 75 pres/abs 3 0.284 Ilex species all 7 75 pres/abs 2 0.243 Ilex species holly only 7 35 pres/abs 2 0.243 Ilex species all 7 75 pres/abs 3 0.243 Angiosperm trees and shrubs all 204 75 pres/abs 2 0.046 Angiosperm trees and shrubs all 204 75 pres/abs 3 0.046 All tree and shrub species all 216 75 pres/abs 2 0.015 All tree and shrub species all 216 75 pres/abs 3 0.015 State of Florida Florida community classifications (50 terrestrial community types) All tree and shrub species all 255 50 pres/abs 2 0.023 All species all 383 50 pres/abs 2 0.057 Notes: Observed and expected correlation coefficients (r) for the relationship between co-occurrence and phylogenetic distance of species pairs are shown. P values (two-tailed test) are determined from a null distribution of 999 randomizations plus the observed value. the Americas, and Cenoud et al. (2000) for Ilex, based results when using presence/absence data, rather than on the Eocene radiation of the genus, not the earliest basal area, and applying null models 2 or 3, although the appearance of extinct basal lineages. Higher level branch results were somewhat less statistically significant (Table lengths, if available, were converted to millions of years, 1). These results are very similar to previous analyses based on the fossil age for the deepest node. This (Cavender-Bares et al. 2004a), despite reduced sampling method of branch length calculation provides only a first and a less resolved phylogeny. approximation of relative evolutionary distances be- Increasing taxonomic inclusiveness.—The effect of tween species. However, given that molecular data could increasing the number of species in the community not be used for branch length estimation, this method is analysis was examined with data sets that included an improvement over using only the tree topology itself. angiosperm trees and shrubs, all trees and shrubs, all recorded angiosperm species, and all recorded plants RESULTS (Table 1). When angiosperm tree and shrub species were included, we found no pattern to the data. This result Plot survey data did not change significantly if we excluded oaks from the Oak species: basal area vs. presence/absence.—Quercus analysis. However, the observed correlation coefficient species showed significant phylogenetic overdispersion became more negative than 818 of the simulated r when we compared pairwise co-occurrence values to values, up from only 650 (Table 1), indicating a shift phylogenetic distances and used null models 1 or 3 with toward greater clustering. When examining all tree and basal area (Table 1, Fig. 2A). We obtained similar shrub species (122 species), again using basal area and July 2006 SCALE AND FLORIDIAN PLANT COMMUNITIES S115

TABLE 1. Extended. (Table 1, Fig. 2B). This pattern was marginally significant if all communities were used in the analysis and became highly significant if communities where oaks r did not occur were excluded from the analysis. One (expected) Obs.Sim P Phylogenetic pattern difference that is apparent between the plot survey and the community classification is that closely related oak species show higher degrees of co-occurrence when the 0.014 8 0.016 overdispersion community classification is used (Fig. 2A, B). This is not 0.004 31 0.064 weak overdispersion surprising, given that defined community types necessar- 0.024 1 0.002 overdispersion ily include all species that regularly occur in them, and 0.019 33 0.066 weak overdispersion 0.025 650 0.700 no pattern they do not take into account spatial distances among 0.006 818 0.364 no pattern individuals or smaller scale environmental variation that 0.009 979 0.042 clustering might occur within community types. Despite this 0.010 977 0.046 clustering 0.005 992 0.016 clustering difference, the overall pattern of phylogenetic over- 0.009 997 0.006 clustering dispersion in the oaks was clear in both analyses. 0.009 578 0.844 no pattern The hollies, although much less diverse, showed a 0.008 990 0.020 clustering similar but only marginally significant pattern of over- 0.014 999 0.002 clustering 0.005 960 0.080 weak clustering dispersion, based on null model 2 only (Fig. 2C), due in large part to the non-co-occurrence of pairs of close relatives, i.e., Ilex cassine and Ilex opaca, as well as Ilex 0.013 45 0.090 weak overdispersion ambigua and Ilex decidua. Note, however, that the 0.028 1 0.002 overdispersion overdispersion pattern was only apparent if community 0.014 9 0.018 overdispersion types that did not include any Ilex species were excluded 0.424 254 0.508 no pattern 0.135 465 0.930 no pattern from the analysis. The pattern was not significant using 0.005 943 0.114 no pattern null model 3. The pines, a much older lineage, showed a 0.029 109 0.218 no pattern somewhat different pattern (Fig. 2D). The two closest 0.062 34 0.068 weak overdispersion 0.005 175 0.350 no pattern relatives, Pinus palustris and Pinus taeda (subsection 0.005 999 0.002 clustering Australes), do not co-occur, similar to the pattern in the 0.038 999 0.002 clustering other two genera. However, at the other extreme, 0.005 998 0.004 clustering 0.026 979 0.042 clustering distantly related pines also do not occur. Pinus clausa, which is in a different subsection of the genus (Contortae) from all of the other pines in the region (section Australes), does not co-occur with any other 0.021 999 0.002 clustering 0.005 999 0.002 clustering Pinus species. Extending the analysis to communities for the entire state of Florida, a clear pattern of phylogenetic clustering emerged (Table 1). At this scale, which either null model 1 or 3, a clear pattern of phylogenetic includes coastal and subtropical communities, there is clustering emerged. A nearly identical result was obtain- an increase in the number and variety of community ed when presence/absence and null models 2 or 3 were types and the degree of environmental heterogeneity used. When all recorded plant species were examined that is encompassed. Correspondingly, the number of (141 species), the pattern of phylogenetic clustering taxa also increases. Acknowledging the incompleteness became even more significant using null model 1. All of the species list, the number of plant species for the recorded angiosperm species (130 species) showed no entire state more than doubles that of the north central pattern. However, upon removal of the 17 oak species Florida communities. The large spatial scale and high from the analysis, the angiosperms were also signifi- environmental heterogeneity, along with the greater cantly clustered. We obtained very comparable results inclusiveness of taxa, leads to a highly significant pattern for the two types of null models (community random- of phylogenetic clustering. izations vs. phylogeny randomizations) (Table 1), although we did not attempt an exhaustive comparison. Trait evolution and trait similarity within communities Analysis of trait evolution for the leaf data and life Community classification data history attributes for the 120-species data set from the In general, similar results were obtained when plot survey showed that all traits examined were community classifications were used, rather than actual significantly conserved (Fig. 3A). A subset of these plot survey data. Based on the community classification, traits also showed high similarity within communities angiosperm trees and shrubs, as well as all trees and (clustering), including specific leaf area for both sun and shrubs, showed significant phylogenetic clustering. Oaks shade leaves, leaf habit, maximum height, leaf perime- still showed a pattern of phylogenetic overdispersion ter-to-area ratio, and cotyledon type. S116 JEANNINE CAVENDER-BARES ET AL. Ecology Special Issue

FIG. 2. Results of tests for phylogenetic overdispersion or clustering within three lineages in north central Florida. (A) There is a higher than expected correlation between pairwise co-occurrence (based on measurements of basal area of species within 55 0.10-ha plots) and phylogenetic distance (measured in millions of years [Ma], based on fossil records) for oak species. Closely related species tend not to co-occur, whereas species from different lineages do tend to co-occur. (B) A similar pattern emerges when the analysis is based on presence/absence of oaks in defined community types (based on community classification data) within six state parks in the same region. The significance of the depicted relationship is dependent on whether the null model only includes communities in which oaks occur, or whether all defined terrestrial communities found in the parks are included. (C) A similar, but less significant pattern is found for the less speciose Ilex genus. (D) The Pinus genus shows a different pattern. Note that in all graphs multiple data points may be superimposed.

In contrast, when the same traits were examined only similarity within communities), such as seed mass and for the oaks, many fewer traits showed conservatism speciﬁc leaf area (particularly for shade leaves), or wood (Fig. 3B). Conserved traits included leaf mass, leaf area, density, acorn maturation time, and leaf lifespan from leaf habit, seed mass, and cotyledon type. None of these the previous analysis. In both the present study and the traits showed high similarity within communities. Other previous analysis, there was a remarkable absence of traits showed a lack of conservatism, which can also be conserved traits that showed clustering (or high sim- interpreted as various degrees of convergence. Maxi- ilarity) within communities. This contrasts sharply with mum height was the most convergent of the traits the pattern seen for all woody plants (Fig. 3A). For the examined in this study. The high convergence results oaks, trait similarity was highest for convergent traits from the fact that in each of the major oak lineages, and lowest for conserved traits. These results indicate including the red oaks, the white oaks, and the live oaks, that the pattern of phylogenetic overdispersion in oak there exist both short and tall species. This trait also communities is generated by the combination of showed very high similarity within communities. A environmental ﬁltering of convergent traits and over- number of other traits were examined in a previous dispersion of conserved traits. study (Cavender-Bares et al. 2004a) and are indicated with small, open circles in Fig. 3B. Relatively convergent DISCUSSION traits from this analysis included maximum hydraulic It has long been recognized that plants can show a conductivity, whole-shoot transpiration rate, vulnerabil- high degree of evolutionary stasis (e.g., Li 1952, Wen ity to cavitation during drought, absolute and relative 1999, Qian and Ricklefs 2004) and niche conservatism growth rates, ability to resprout from rhizomes, and (Webb et al. 2002, Ackerly 2003, Reich et al. 2003). At bark thickness. At the same time, traits that were fairly the same time, evolutionary processes that cause differ- conserved tended to show overdispersion (or low entiation of sister taxa, such as character displacement July 2006 SCALE AND FLORIDIAN PLANT COMMUNITIES S117

FIG. 3. Conservatism of traits in relation to trait similarity within communities for (A) all woody species and (B) oak species only. Traits are ordered on the x- and y-axes by the ranking of the observed similarity or phylogenetic signal of each trait relative to 999 simulations in the null model (the ranking of the r values of observed data for trait similarity relative to simulated data increases with overdispersion [vs. clustering], and the ranking of the observed phylogenetic signal relative to simulated data decreases with conservatism [vs. convergence]). Traits include the following (labeled solid circles): leaf area, speciﬁc leaf area (SLA)- sun, SLA-shade (SLA-sh), perimeter-to-area ratio (P/A), leaf habit, leaf mass, lobing, petiole length, cotyledon type, seed mass, and height. Open circles represent a suite of other morphological, physiological, and life history traits, measured from trees in the ﬁeld as well as from seedlings in a common garden from a previous study (Cavender-Bares et al. 2004). The contrasting patterns of trait evolution and community similarity for the whole woody plant community and the oak lineage help explain the contrasting phylogenetic structures of communities observed at these different taxonomic scales.

(e.g., Schluter 2000) and adaptive radiation (e.g., similar cannot coexist (e.g., Elton 1946, MacArthur and Givnish et al. 2000) are well documented. To the extent Levins 1967, Chesson 2000). In this study, we suggest a that evolutionary stasis predominates, close relatives are solution for this paradox by showing that plant likely to occur in similar habitats, given that plants will communities can simultaneously exhibit niche conserva- track environments for which they are adapted (Ackerly tism and overdispersion of close relatives. The signal 2003). The principle of competitive exclusion presents a that dominates depends on the scale at which commun- paradox, however, because close relatives that are too ities and the taxa in them are examined. S118 JEANNINE CAVENDER-BARES ET AL. Ecology Special Issue

clustering occurs at larger spatial scales when a greater range of community types is included (Fig. 4). The phylogenetic structure of communities depends even more strikingly on the phylogenetic scale, or inclusiveness of taxa. Communities within a region may appear overdispersed at one phylogenetic scale (e.g., within one lineage), but clustered at a broader phylogenetic scale (e.g., when including all seed plants in the community). This allows a reconciliation of apparently contradictory results, as both niche conservatism and species interactions are important forces in community assembly, but are dominant at different scales. Species interactions that give rise to phylogenetic overdispersion show a stronger signal when communities are delimited to include a single monophyletic lineage. Niche conservatism shows a stronger signal the more broadly a community is defined taxonomically. Speciose lineages, such as the oaks, may be more likely to show overdispersion than less speciose lineages if, for example, increased diversity leads to increased competition among closely related species. Phylogenetic patterns are also likely to be lineage- specific, and it is not known the extent to which the oaks represent an exceptional scenario. Hence, the specific results found here might not be generalizable to other systems (see, for example, Kembel and Hubbell 2006).

FIG. 4. The phylogenetic structure of communities is Nevertheless, the study does demonstrate the potential of dependent on the scale at which they are defined. Apparent phylogenetic patterns to vary predictably with scale and overdispersion within forest communities (A), for example, can demonstrates that both phylogenetic overdispersion and appear as clustering when grassland and wetland communities clustering can occur at different spatial and taxonomic are included (B). At the same time, phylogenetic structure also depends on the phylogenetic scale or taxonomic inclusiveness. scales in the same study system. Apparent overdispersion within a lineage is more likely to appear as a dominant pattern of clustering when the analysis Phylogenetic structure within single lineage communities includes many lineages. Phylogenetic dispersion patterns of species in communities that are narrowly defined taxonomically appear Phylogenetic structure of communities at different to be lineage specific, and may depend on intrinsic spatial and taxonomic scales properties of lineages, biogeographic history, and lineage diversity. One possibility is that phylogenetic Both the spatial scale and resolution of species overdispersion might be more likely in speciose lineages abundance data influence the observed phylogenetic due to intensification of species interactions. In addition, structure in community assemblages. Greater over- overdispersion might be more likely in lineages that have dispersion is detected at smaller spatial scales and with undergone adaptive radiations more or less in situ, as higher resolution data. This is demonstrated by more the evolutionary process should result in close relatives significant overdispersion when using basal area within occupying different habitats as well as high local plots rather than presence/absence (Table 1). Basal area diversity. The American oaks are a likely example of provides higher resolution for species distributions, and such a radiation. Oaks are believed to have reached the the extent to which they overlap within communities, Americas from Eurasia ;40 million years ago and are and hence could provide more precision for detecting subsequently thought to have undergone a rapid overdispersion. The difference in null model is appar- radiation with all of the major subgenera appearing in ently not a factor in this outcome, as the same results the fossil record by 35 million years ago (Crepet and were found using null model 3 (randomization of the Nixon 1989). Many of the southeastern oaks are phylogeny instead of the communities; Table 1). endemic to the region, and biogeographic evidence Phylogenetic overdispersion is more easily detected suggests that diversification of the oaks in the Americas among close relatives when the communities included occurred in their current localities (Manos et al. 1999, in the analysis are limited to only those in which the Manos and Stanford 2001). The Florida peninsula was focal lineage occurs (Table 1). This effectively reduces not connected with North America and was submerged the spatial area and environmental heterogeneity exam- under the ocean until 20 million years ago (Webb 1990). ined. These results make clear that phylogenetic over- The colonization of Florida, therefore, occurred sub- dispersion can occur at small spatial scales, even while sequent to this time period. Fossil records indicate that July 2006 SCALE AND FLORIDIAN PLANT COMMUNITIES S119 the flora was originally tropical, but increasingly ultimately depends on (1) the evolutionary history of invaded by deciduous hammock communities, similar species’ traits and (2) the extent to which traits influence to those of today, starting about 17 million years ago as species distributions across environmental gradients or the climate cooled. The availability of new habitats for prevent coexistence due to species interactions. Closely colonization might have facilitated rapid adaptation, related species are likely to co-occur if traits important possibly aided by promiscuous exchange of genetic for environmental filtering are conserved. On the other material among species. The present-day overdispersion hand, if traits important for environmental filtering are of oaks (Table 1; Mohler 1990, Cavender-Bares et al. convergent, or many different strategies for existing in a 2004a) could thus be the result of their evolutionary given habitat are possible, then closely related species history of adaptive radiation into novel habitats. might not co-occur. Likewise, if similarity in particular The hollies show some degree of overdispersion traits prevent coexistence, and such traits are conserved, (Fig. 3C) under null model 2, although this pattern is then closely related species are again unlikely to co-occur. not significant under null model 3, and hence the signal is Of course, if species distributions are not related to somewhat ambiguous. Ilex diversified in the Eocene, ;50 their phenotypes, as predicted by neutral models (Hub- million years ago and was already dispersed over all of bell 2001), then phylogenetic patterns in community the major continents by that time (Cuenoud et al. 2000). structure are unlikely to emerge (Kembel and Hubbell These small-seeded, bird-dispersed species arrived in 2006). However, lack of clear phylogenetic patterns Florida over the course of the past 20 million years, but might also result from species interactions and environ- for the most part evolved before then on other continents mental filtering operating in opposing directions. In this in other contexts. With the exception of two pairs, Ilex study, for example, phylogenetic overdispersion of oaks cassine and Ilex myrtifolia, and Ilex decidua and Ilex apparently masks the pattern of phylogenetic clustering ambigua, speciation did not occur in the context of the that predominates among angiosperm lineages. When other hollies that currently inhabit Florida, and currently oaks are removed from the analysis, the clustering sympatric congeners could have large intervening branch pattern becomes apparent (Table 1). length distances (Cuenoud et al. 2000). The lack of co- Among species in the broadly defined Floridian occurrence between the closely related members in each communities, all traits examined showed a fairly high pair gives rise to the apparent trend of overdispersion degree of conservatism (Fig. 3A). A subset of these traits under null model 2 (Fig. 3C). The relatively weak signal, showed high similarity within communities (clustering), however, might result from a very different biogeo- including specific leaf area for both sun and shade graphic history of the hollies or, alternatively, from the leaves, leaf habit, maximum height, leaf perimeter-to- fact that the hollies are much less speciose than the oaks, area ratio, and cotyledon type. These traits have particularly in this study region. generally been shown to be important in the ability of The pines show a hump-shaped phylogenetic pattern, species to respond to abiotic stress factors and to in which only pines of intermediate relatedness co-occur influence species distributions across environmental (Fig. 3D). It has previously been noted for the pines in gradients, critical evidence for their potential role in Florida that sister species do not co-occur and that co- environmental filtering. For example, specific leaf area occurring species are not closely related (Adams and and leaf habit, a proxy for leaf lifespan, are both Jackson 1997). Hence, some degree of phylogenetic important in the carbon economy of plants (e.g., overdispersion has been observed within the genus, even Kikuzawa 1991, Damesin et al. 1998, Kikuzawa and though we did not detect it in north central Florida. The Ackerly 1999) and have been well documented to vary lack of clear phylogenetic signal in pine communities with soil nutrient availability at various spatial scales might result from the lower diversity of the pines relative (Monk 1966, Reich et al. 1999, Wright et al. 2002, Reich to the oaks, causing congeneric competition to be less et al. 2003). Specific leaf area is also likely to be important among the pines. associated with canopy openness and soil moisture The strong signal of overdispersion in the oaks might availability (Wright et al. 2002). Maximum height has result from their history of adaptive radiation and their been linked to growth rate (Thomas 1996), and taller high local diversity. Additional studies are needed to height is a competitive strategy for accessing light in determine (1) the extent to which overdispersion among productive environments (Tilman 1988). In lower close relatives depends on historical and lineage-specific productivity environments or in communities prone to factors and (2) the extent to which local diversity within burning, less investment in aboveground biomass is lineages might influence the intensity of species inter- expected (Schwilk and Ackerly 2001, Cavender-Bares et actions that give rise to phylogenetic overdispersion in al. 2004b). Leaf perimeter-to-area ratio has been communities. correlated to hydraulic conductance (Sack et al. 2003) and is likely to be important in distribution patterns Trait evolution and community assembly across soil moisture gradients (Brodribb and Holbrook Nonneutral ecological processes that influence com- 2003). The primary role of cotyledons as either storage munity assembly act on the phenotypes of species. organs or photosynthetic organs represents a trade-off Therefore, the phylogenetic structure of communities in regeneration strategies that depend on resource S120 JEANNINE CAVENDER-BARES ET AL. Ecology Special Issue availability (Kitajima and Fenner 2000). The previously oaks (;60-fold difference) as it does across all species established linkages to resource capture and use for all examined at the broader scale, but this level of variation of the traits that show high similarity (clustering) within within a clade is unique to the oaks in our study. The communities support the interpretation that these traits shift in patterns of trait evolution toward increasing are likely to be important for habitat tracking and conservatism at broader taxonomic scales is likely to environmental filtering. Clustering of conserved traits is explain the concomitant shift in the phylogenetic critical in explaining the emergent pattern of phyloge- structure of communities toward clustering. It also lends netic clustering in Floridian plant communities. support to the view that niche conservatism is wide- Other traits, including leaf mass, leaf area, lobedness, spread among plants (Wen 1999, Webb et al. 2002, petiole length, and seed mass showed low similarity Ackerly 2003, Qian and Ricklefs 2004). within communities. These traits might be less adaptive The oaks, which dominate many woody communities to environmental factors, or at least to those that vary at in north central Florida, appear to represent a special the same scale as the plot survey, and might be case in this study system. They show an unusual amount associated with architectural constraints. Alternatively, of lability in certain functional traits, such as maximum there could be multiple trait strategies that are successful height, water transport capacity, growth rate, and the in a given community. For example, leaf size and leaf ability to resprout from rhizomes (Cavender-Bares et al. lobing have been linked to boundary layer conductance 2004a), traits important for habitat specialization and heat load (Givnish 1976), which could be important (Cavender-Bares et al. 2004b). The observed phyloge- in environmental filtering. However, various combina- netic overdispersion among the Floridian oaks could be tions of leaf size, shape, leaf angle, leaf display, and a result of their history of adaptive radiation and might pubescence can achieve a similar energy balance be maintained by reduced competition among co- (Lambers et al. 1998, Ackerly 1999). Leaf size, seed occurring species of different subgenera or lower size, and petiole length might also be more related to density-dependent mortality (Cavender-Bares et al. plant architecture than to external environmental 2004a). The latter possibility has been hypothesized as gradients (e.g., Mazer 1989, Lord et al. 1995, Moles et a result of pathogen specificity at taxonomic levels above al. 2005), and these traits have not generally shown the species (Janzen 1970, Webb and Gilbert 2006). At strong trait–environment correlations. larger taxonomic and spatial scales, however, Floridian While all the traits measured in this study were fairly plant communities show phylogenetic clustering. This conserved in the broad analysis, many of the same traits can be accounted for by conservatism of functional tended to be convergent, or not different from random traits that influence species distributions. These con- expectation, within the oak genus (Fig. 3B). For trasting patterns that emerge within the same study example, maximum height, the perimeter-to-area rela- system illustrate the importance of scale in detecting tionship, and specific leaf area were more convergent opposing ecological and evolutionary forces. within the oaks than among the larger species pool. This result is not surprising if trait variance among lineages is ACKNOWLEDGMENTS higher than the variance within a lineage (Felsenstein J. Cavender-Bares would like to thank Cam Webb and 1985, Harvey and Pagel 1991). In other words, even if Jonothan Losos for the invitation to participate in this special sister taxa show divergent morphology, recent common issue. We thank Kelly McPhearson and Anne Barkdoll at the ancestry of closely related species is likely to limit the District 2 Park Service for providing species lists and community data maps for the six state parks in north central amount of divergence within a lineage, relative to that Florida. Ginger Morgan is acknowledged for facilitating found among distantly related lineages. This could permits. George Otto, Tony Davanzo, and Michael Stevens explain the shift to conservatism at broader taxonomic are gratefully acknowledged for resampling 32 of the permanent scales. Specific leaf area, a fairly labile trait within the plots. Elizabeth Salisbury collected and sent to us all of the oaks, for example, varied roughly threefold within the leaves for the leaf trait analysis, and Marjorie May Haight helped scan the leaves. We thank Doug Hornbeck, Sue Mauk, genus, but exceeded fivefold among all species sampled. Kent Cavender-Bares, David Ackerly, Jason Teisinger, Hannah However, in a study of almost 13 000 seed plants, Moles Nendick-Mason, Erik Smith, and Park Manager Randy Brown et al. (2005) showed that higher divergences sometimes for logistical and other support or assistance with various occurred within a family than between families. A aspects of the work in Florida. Anurag Agrawal and two number of genera, particularly speciose genera in which anonymous reviewers are gratefully acknowledged for valuable comments on the manuscript. many members co-occur, have also been shown to have divergences within them equal to or greater than those LITERATURE CITED between co-occurring species that are distantly related Ackerly, D. 1999. Self-shading, carbon gain and leaf dynamics: (Silvertown et al. 2006). Therefore, the shift toward trait a test of alternative optimality models. Oecologia 119:300– conservatism at the broader phylogenetic scale can also 310. be explained as a result of swamping out the signal of Ackerly, D. D. 2000. Taxon sampling, correlated evolution, and independent contrasts. Evolution 54:1480–1492. high trait lability within the oaks by the addition of Ackerly, D. D. 2003. Community assembly, niche conservatism many more taxa that have conserved traits. Maximum and adaptive evolution in changing environments. 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APPENDIX A Species used in each analysis (Ecological Archives E087-114-A1).

APPENDIX B References for phylogenetic data (Ecological Archives E087-114-A2).

APPENDIX C Phylogenetic trees used in each analysis (Ecological Archives E087-114-A3). Ecology, 87(7) Supplement, 2006, pp. S123–S131 Ó 2006 by the Ecological Society of America

PHYLODIVERSITY-DEPENDENT SEEDLING MORTALITY, SIZE STRUCTURE, AND DISEASE IN A BORNEAN RAIN FOREST

1,3 2 1 CAMPBELL O. WEBB, GREGORY S. GILBERT, AND MICHAEL J. DONOGHUE 1Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut 06520 USA 2Environmental Studies Department, University of California-Santa Cruz, California 95064 USA

Abstract. Density-dependent models that partition neighbors into conspecifics and heterospecifics ignore the great variation in effect of heterospecifics on focal plants. Both evolutionary theory and empirical results suggest that the negative effect of other plants on a focal plant should be higher for closely related neighbors than for less related neighbors. Using community-wide seedling mortality data from a forest where density dependence has previously been found, we searched for significant phylogenetic neighborhood effects (the ‘‘phylodiversity’’ neighborhood) on seedling (,50 cm tall) survival at various spatial scales. Logistic regression models were used, with 19-mo survival of individual seedlings as the response. We found a significant positive effect of nearest taxon phylodiversity on seedling survival at the 36-m2 scale and the 4-m2 scale, indicating that seedling survival is enhanced by being in a neighborhood where heterospecifics are not closely related. At all scales there was a strong negative effect of conspecific seedling density on focal survival, and at small scales there was also an effect of heterospecific density, indicating generalized competition. We place these results (for seedling dynamics over a relatively short period of time) in the context of changes in phylodiversity between different size classes of plants in the same forest, which integrate the effects of dynamics of all size classes over long time periods. At the 36-m2 scale, there was an increase in nearest taxon phylodiversity (i.e., a decrease in phylogenetic clustering) from the seedlings (,50 cm tall) to the poles (1–5 cm diameter), consistent with the positive effect of local phylodiversity on seedling survival. In contrast, there was a marked decrease in average phylodiversity from seedlings to saplings at the same scale. The trees in the 1600 m2 surrounding the seedling plots had much lower phylodiversity than either the seedlings or saplings. Taken together, these results suggest that (1) over short time and spatial scales, local seedling phylodiversity has a positive effect on seedling survival, possibly via interaction with pathogens (which we discuss in detail), but (2) over longer time periods and larger spatial scales the effect of abiotic-related mortality results in habitat filtering for phylogenetically conserved traits. Key words: Borneo; community phylogenetic structure; density dependence; phylodiversity; plant pathogen infection; seedlings.

INTRODUCTION The strength of conspecific density (or interplant In both theoretical and empirical studies, intraspecific distance), relative to the effect of overall competitive negative density dependence has been shown to be pressure, can be measured by including the density (or important for slowing competitive exclusion and main- distance) of heterospecifics as a separate factor in taining diversity in rain forest trees (Wright 2002). Most models (Connell et al. 1984, Uriarte et al. 2004). analyses focus on the statistical effects of conspecific However, simply dividing species into conspecifics and density on either focal-plant performance or species heterospecifics obscures the great variation in effects of demographic parameters (Martinez-Ramos et al. 1988, different species on any focal species (Pacala et al. 1996). Condit et al. 1994, Harms et al. 2000). In some cases, the One solution is to give separate ‘‘competition’’ param- effect of distance from focal plant to conspecifics has been eters to each pairwise interaction (Canham et al. 2004, used as a correlate of plant density (Hubbell 1980, Connell 2006). Unfortunately, a great deal of data is required to et al. 1984, Hubbell et al. 1990, Gilbert et al. 1994). fit a model with so many free parameters. If the effect of one species on another was random with respect to the Manuscript received 26 January 2005; revised 18 July 2005; plants’ phenotypes, there would be no solution other accepted 21 July 2005; final version received 9 September 2005. than this multiparameter modeling. However, interspe- Corresponding Editor: A. A. Agrawal. For reprints of this cific interactions are influenced by the anatomical and Special Issue, see footnote 1, p. S1. 3 Present affiliation: Arnold Arboretum of Harvard Uni- physiological similarity of the species involved, and versity. E-mail: [email protected] gross similarity is not distributed at random among S123 S124 CAMPBELL O. WEBB ET AL. Ecology Special Issue

PLATE 1. The Air Putih River as it passes through granite hill forest at the Cabang Panti study area, Gunung Palung National Park, Indonesia. Photo Credit: C. Webb. organisms; it is generally highest among taxa that share for the Rhizophoraceae). Additionally, higher taxonom- a recent ancestor (Felsenstein 1985, Harvey and Pagel ic groups defined using morphological (e.g., floral) 1991). In general, we expect greater negative interactions characters might have little ecological coherence; either among individuals that are more phenotypically similar, more- or less-inclusive clades than named-rank clades be they competing for a similar vector of resources or might be more ecologically meaningful classes. With the negatively affecting each other via shared seed preda- great progress made recently in resolving the phyloge- tors. Thus, we expect that closely related taxa should netic relationships among organisms, we can now move have a greater negative influence on each other than taxa beyond ranks (such as family and genus) to use that are more distantly related. Indeed, Uriarte and phylogenetic distances (e.g., age) among taxa. In this colleagues (2004) recently analyzed neighbor-dependent paper, we include such a phylogenetic distance factor in sapling growth at Barro Colorado Island and found that the analysis of community seedling mortality in lowland the negative effect of a neighbor on a target was greater rain forest at Gunung Palung in Indonesian Borneo (see when both plants were in the same taxonomic family. Plate 1; Webb and Peart 2000). At this site, density- Adding a relatedness parameter to models of density dependent seedling mortality has previously been found dependence might therefore increase the predictive both in single species (Webb and Peart 1999, Blundell power of these models significantly, at the expense of and Peart 2004) and as a community-wide compensatory just a few degrees of freedom. trend (Webb and Peart 1999). The development of phylogenetic methods over the We take two approaches toward testing the contribu- past few decades has exposed the nonequivalence of taxa tion of the phylogenetic relatedness structure of assigned to the same traditional rank. For example, in a neighbors (hereafter the neighborhood ‘‘phylodiversity’’; recent supertree study of the relationships among cf. Faith 1992) to seedling dynamics. First, we examine angiosperms (Wikstro¨m et al. 2001), the estimated its effect on the survival of seedlings in small quadrats in minimum age of family clades varied over an order of multifactorial models that also include the effect of magnitude (e.g., 108 Ma for the Aristolochiaceae, 9 Ma conspecific density and heterospecific density. The July 2006 PHILODIVERSITY AND SEEDLING DYNAMICS S125 period chosen for the analysis is the same as that for direct age estimates are not available. Because many of which community-wide density dependence was found the genera in the plot have yet to be sampled in a (Webb and Peart 1999). Second, we analyze changes in phylogenetic study, many of the families were modeled local phylogenetic structure with plant size class. Any in the final supertree as a polytomy, and all the genera influence of phylodiversity on seedling survival (a were polytomies. This means that the significant dynamic measure) should eventually leave an imprint phylogenetic results reported here are not dependent on the phylogenetic structure of surviving plants (a static on, for example, phylogenetic conservatism in pathogen pattern). If, for example, mortality is higher in species host use only among sibling species. Instead, they must that are sharing a neighborhood with closely related reflect patterns of conservatism that extend at least to species than in species that are phylogenetically isolated the root of genera nodes, and possibly beyond. It is from their neighbors, then this should be observed as an likely that some ecologically relevant characters are even increase in phylodiversity (and a decrease in net conserved at family nodes and above. relatedness) of plants of increasing size. Alternatively, For the analysis of seedling dynamics, we used if habitat filtering leads to strong phenotypic and measures of survival of all individuals of all seedling phylogenetic attraction (Webb et al. 2002, Cavender- species (5–50 cm tall) over a 27-month period from Bares et al. 2004), the plants in increasingly taller size March 1994, in a subset of 12 of the plots (n ¼ 2296 classes should show decreased phylodiversity. seedlings total). This subset included only plots with We thus ask the following questions: (1) Does adding completed seedling identification as of November 1994, a phylogenetic neighborhood (phylodiversity) term and was a sample of all 28 plots stratified by elevation increase the fit of density-driven models of seedling and subhabitat. Individual plants were mapped to a mortality? (2) Does the influence of neighborhood 0.25-m2 quadrat within the 36-m2 seedling plot, allowing phylodiversity vary with neighborhood scale? (3) Are analysis in nested square neighborhoods of 0.25, 1, 4, changes in the (static) phylogenetic structure with and 36 m2 (the design of the seedling plots included two increasing size classes consistent with phylodiversity- 1-m walkways, which precluded using neighborhoods dependent seedling mortality? between 4 m2 and 36 m2; see plot diagram in Webb and Finally, we develop a mechanistic hypothesis for the Peart [1999]). We first counted the total seedling number mediation of these phylodiversity effects by pathogens, per quadrat (for each sized quadrat: 0.25, 1, 4, and 36 the most likely causal agents of density dependence in m2) at the beginning of the census period, and these forests. determined whether each individual was alive or dead at the end of 27 months. We also calculated the number METHODS of conspecific individuals and the relative phylodiversity 2 In 1993, Webb set up 28 36-m seedling plots in of each species in the quadrat. Phylogenetic diversity lowland rain forest in the Gunung Palung National (Faith 1992), or phylodiversity, is negatively related to Park, West Kalimantan (Indonesian Borneo; site loca- the extent of phylogenetic clustering in a sample (Webb tion: 1.21258 S, 110.10788 E; see Plate 1). The height of 2000, Webb et al. 2002, Cavender-Bares et al. 2004), and all woody plants was measured for plants taller than 5 positively related to measures of phylogenetic distance cm and ,1.0 cm dbh (diameter at breast height [1.3 m among taxa in a sample. We measured relative high]), and dbh was measured for plants 1.0–5.0 cm dbh. phylodiversity in two ways, (1) using the mean Each seedling plot was nested inside a 40 3 40 m tree phylogenetic distance (in units of millions of years) plot in which all trees .10.0 cm dbh were measured and from the species of the focal individual to all other (n – identified (see Webb and Peart [1999, 2000] for further 1) species in the quadrat, and (2) using the minimum details). phylogenetic distance to any heterospecific species in the We constructed a single phylogeny that included all quadrat (i.e., to the nearest taxon, or taxa, where several plant species occurring in any of the plots. We first ran were equidistant). Both distances were then standardized the species list through the online tool Phylomatic by the mean expected phylogenetic distance, given the (Chazdon et al. 2003, Webb and Donoghue 2005) to number of species in the quadrat, in order to correct for produce a tree topology based on the Angiosperm the effect of sample species richness; details of the same Phylogeny Group (APG) II backbone (APG 2003; P. F. standardization for the indices of phylogenetic cluster- Stevens, Angiosperm Phylogency Website, version 6, 4 ing, net relatedness index (NRI) and nearest taxon index (available online), using the Phylomatic reference (NTI), are given in Webb et al. (2002). We refer to the megatree R20040402. We then used the BLADJ resulting metrics as relative average phylodiversity, algorithm of Phylocom (Webb et al. 2004, Moles et al. APd0, and relative nearest taxon phylodiversity, NTPd0 2005) to constrain the internal nodes of the tree to the (the prime indicates that the metric is relative to the age estimates of Wikstro¨m et al. (2001). The algorithm identity of focal species; absolute APd and NTPd are then interpolates the other nodes of the tree for which used in analysis of size class; and ‘‘Pd’’ is used to differentiate from Faith’s [1992] ‘‘PD,’’ which is 4 hhttp://www.mobot.org/MOBOT/research/APwebi measured differently). Phylodiversity is oriented here S126 CAMPBELL O. WEBB ET AL. Ecology Special Issue so that larger positive values of phylodiversity indicate (mean 6 SE) was 209 6 22, 48.4 6 4.4, 12.3 6 0.63, and communities whose species are less closely related. 3.66 6 0.11; the number of species per quadrat was 48.0 We modeled survival of each seedling (categorical 6 3.5, 18.8 6 1.1, 7.46 6 0.29, and 2.88 6 0.072; the data: lived vs. died) using four separate logistic mean number of APG family clades per quadrat was regressions: (1) survival as a function of the total 23.1 6 1.1, 12.5 6 0.52, 5.97 6 0.19, and 2.70 6 0.061; number of seedlings in the same quadrat, (2) survival the mean number of individuals per species per quadrat as a function of the number of conspecifics in the same was 4.36 6 0.41, 2.56 6 0.13, 1.65 6 0.045, and 1.27 6 quadrat and the number of heterospecifics in the same 0.018. quadrat, (3) survival as a function of the number of A total of 20 models were fit (Table 1), and, in all conspecifics in the same quadrat, and (4) survival as a models in which it was present as a factor, the function of the number of conspecifics in the same (logarithm of the) number of conspecific individuals quadrat, number of heterospecifics in the same quadrat, was the most predictive factor for seedling survival. At and relative phylodiversity of the community in the quadrat sizes of 4m2, seedling survival was strongly same quadrat. The total number of species in the negatively related to (the logarithm of) total seedling quadrat was included in preliminary analyses, but was density. Heterospecific (log of) seedling density was never a significant effect, and was dropped from final negatively related to survival only at the smallest models. quadrat size (0.25 m2), at which scale the effect of total For the static size class analysis, we used identity and density was also strongest. Relative nearest taxon height for all species alive in June 1996 in the full set of phylodiversity (NTPd0) was positively associated with 28 seedling and tree plots. Based on plant size at that survival at the 36-m2 and 4-m2 scales. At both scales, the time, we assigned plants to four size classes: seedlings (0– addition of the NTPd0 phylodiversity term provided the 50 cm tall), saplings (50 cm tall to 1 cm dbh), poles (1–5 best overall model fit, as measured by Akaike’s cm dbh), and trees (.10 cm dbh). A total of 389 species Information Criterion (AIC). Relative average phylodi- were recorded as seedlings, 277 as saplings, 161 as poles, versity (APd0) was negatively correlated with seedling and 325 species as trees. We drew up a species list for survival at the largest (36-m2) scale. each size class in each of the 28 plot locations, and this was passed through Phylocom (Webb et al. 2004) to Change in phylodiversity with increasing size class calculate the net relatedness index and the nearest taxon From seedlings to poles, average phylodiversity (APd) index (Webb et al. 2002) of these species lists on the decreased and nearest taxon phylodiversity (NTPd) phylogeny of the whole pool of 548 species (see the increased, while phylodiversity declined from seedlings Supplement). We recognize that not all taxa in the pool to trees for both measures (Fig. 1). The influence of the can occur in every size class (e.g., shrub and liana taxa four size classes on phylodiversity was significant overall will not be found in the tree plot samples), but we use a (Kruskall-Wallis rank-sum test; APd, v2 ¼ 24.1, df ¼ 3, P common phylogeny because measures of phylogenetic ¼ 2.3 3 105; NTPd, v2 ¼ 34.7, df ¼ 3, P ¼ 1.4 3 107), structure (NRI, NTI) are directly comparable only and the following pairwise comparisons were significant within a common pool phylogeny. We do not attempt using Wilcoxon rank-sum tests: APd, seedling vs. to test whether the indices are different from zero sapling (W ¼ 209, P ¼ 0.0023), seedling vs. pole (W ¼ (indicating either phylogenetic clustering or evenness; 257, P ¼ 0.026), seedling vs. tree (W ¼ 130, P ¼ 6.5 3 Webb 2000), because the null model of random 106), pole vs. tree (W ¼ 195, P ¼ 0.0010); NTPd, sampling from the pool would be inappropriate. Because seedling vs. tree (W ¼ 207, P ¼ 0.002), sapling vs. tree (W we are using estimates of absolute age between taxa, the ¼ 131, P ¼ 7.2 3 106), pole vs. tree (W ¼ 31, P ¼ 9.2 effect of taxon sampling on phylogenetic distance is 31012). In a separate analysis, lists of seedling and largely removed. sapling species were made for the 252 quadrats of 4 m2: The indices NRI and NTI were multiplied by negative both APd and NTPd decreased from seedlings to one to obtain measures of phylodiversity (average, APd, saplings (APd, v2 ¼ 7.17, df ¼ 1, P ¼ 0.0073; NTPd, v2 and nearest taxon, NTPd, respectively), directly com- ¼ 10.3, df ¼ 1, P ¼ 0.0012). parable to those used in the analysis we have described of seedling mortality. Median values of phylodiversity DISCUSSION for the 28 plots were compared across size classes. We Our analysis of community-dependent seedling sur- conducted all analyses for both subprojects using the vival suggests that at larger scales (4 m2 and 36 m2) there statistical language R (R Project 2004) and Phylocom is a significant beneficial effect of local phylodiversity (Webb et al. 2004). (NTPd): that is, the chance that a seedling survives

RESULTS increases if surrounding plants are not closely related to it, even after the effects of conspeciﬁc and heterospeciﬁc Phylodiversity and seedling survival density have been removed. There was no such effect at A summary of the variation in numbers of individuals smaller scales. At the same scale of 36 m2, we also and species follows: for quadrats of 36, 4, 1, and 0.25 m2, observed an increase in phylodiversity from the seedling respectively, the number of individuals per quadrat to the sapling to the pole size classes, when measured as July 2006 PHILODIVERSITY AND SEEDLING DYNAMICS S127

TABLE 1. Effect of local seedling density and phylodiversity on seedling survival, for four quadrat sizes. Five logistic models were ﬁt for each of the four quadrat sizes: 0.25, 1, 4, and 36 m2.

Significance of factor Log Log Log Phylodiversity Phylodiversity Quadrat size and model AIC df (total N) (conspecific N) (heterospecific N) (APd0) (NTPd0) 36 m2 Total density 3138 2295 NS Partitioned density 3114 2294 0.11*** þ0.28* Conspecifics only 3118 2295 0.13*** Complete (APd0) 3103 2293 0.15*** þ0.25* 0.25* Complete (NTPd0) 3101 2293 0.085** þ0.25* þ0.22** 4m2 Total density 3129 2295 0.21** Partitioned density 3114 2294 0.17*** NS Conspecifics only 3112 2295 0.18*** 0 Complete (APd ) 3113 2293 0.19*** NS NS 0 Complete (NTPd ) 3111 2293 0.14** NS þ0.13** 1m2 Total density 3123 2291 0.20** Partitioned density 3109 2290 0.23*** NS Conspecifics only 3107 2291 0.24*** 0 Complete (APd ) 3110 2289 0.25*** NS NS 0 Complete (NTPd ) 3110 2291 0.21** NS NS 0.25 m2 Total density 2838 2093 0.34** Partitioned density 2834 2092 0.31*** 0.18* Conspecifics only 2838 2093 0.33*** 0 Complete (APd ) 2835 2827 0.33*** 0.18* NS 0 Complete (NTPd ) 2837 2091 0.33** 0.17* NS Notes: Survival is measured as a positive response, so a negative parameter estimate indicates a negative relationship of the factor with seedling survival. Phylodiversity increases with decreasing relatedness of the focal taxon to the other taxa in the sample. Akaike’s Information Criterion (AIC) measures the complexity of the fitted model; a lower value indicates a better fit of the model to the data. Key to abbreviations: APd0, relative average phylodiversity; NTPd0, relative nearest taxon phylodiversity. *P ¼ 0.05; **P ¼ 0.01; ***P ¼ 0.001. nearest taxon phylodiversity, NTPd. These two results which removes the main effect of sample species richness are consistent with each other: if survival is higher for on phylogenetic relatedness. Additionally, there was no seedlings that are more distantly related, then as the significant relationship of plot species richness with APd cohort ages the overall net relatedness should decrease, or NTPd within any size class (8 tests, n ¼ 28 plots). as closely related taxa are ‘‘weeded out.’’ However, a However, thorough simulation studies to explore the contrasting pattern is observed in the measure of average behavior of these metrics are desirable and are underway phylodiversity: at the 36-m2 scale, there is an apparent (S. Kembel, personal communication; N. Kraft, personal detrimental effect of APd on seedling survival, and a communication). consistent decrease in APd phylodiversity is observed in Instead, we believe that the observed patterns result the static data, from seedlings to saplings, and from from the different aspects of phylogenetic structure seedlings to poles. We also observed that for both NTPd captured by the two measures of phylodiversity. Imagine and APd measures, there was a large decrease in that the seeds arriving at a particular site are a random phylodiversity from seedlings to trees, i.e., tree species sample of all the plants in a forest, plants that occupy a were far more clustered phylogenetically than were number of habitats. Imagine also that some large clades seedling species. Previous work showed trees in a plot of many taxa possess characters that will increase to be more phylogenetically clumped than expected by survival in this particular habitat, while other clades chance (Webb 2000). do not have these characters. Over time, there should be How can we reconcile these different results? When a net increase in average relatedness of the survivors at a comparing different size classes for the same area, we site (i.e., a decrease in APd). However, if negative must be aware that the different number of individuals in interactions are highest among taxa that are very closely the different size classes, and thus the different expected related (sister species, or consectionals), there may number of species, could be leading to an artifactual simultaneously be a reduction over time in the phylo- change in metrics. While this remains a possibility, we genetic distance to the most closely related surviving feel it is not the cause of the patterns we observe, because species (i.e., an increase in NTPd). All that is required the standardization of the indices uses the expected value for these two apparently opposite changes to occur of relatedness for a given n species (Webb et al. 2002), simultaneously is that the characters for habitat filtering S128 CAMPBELL O. WEBB ET AL. Ecology Special Issue

become trees, nearest taxon phylodiversity (NTPd) must also eventually decrease, as the only survivors will belong to numerous ‘‘clumps’’ of related taxa. While this explanation surely oversimplifies the phylogenetic distribution of characters necessary for survival, we believe it helps explain the observed patterns. The large decrease of average and nearest taxon phylodiversity in trees is expected because of both the larger spatial scale of the tree plots and the length of time over which processes have operated as seedlings grow to trees. During this time, the effects of habitat filtering have dominated change in both measures of phylodiversity. We note that it is the combination of short-term dynamic data and static data reflecting long-term outcomes that provides these insights into community dynamics.

A framework for the influence of pathogens on phylodiversity As at other tropical rain forest sites (e.g., Augspurger 1984), available evidence points to pathogens being the primary cause of density-dependent mortality at Gu- nung Palung (Webb and Peart 1999). Pathogens have been shown to strongly influence plant community structure and diversity in a number of natural and agricultural plant systems (see review by Gilbert [2002]). To the extent that pathogens are more likely to cross- infect closely related hosts (Parker and Gilbert 2004), negative interactions between plant species should be strongest among very close relatives (Mack 1996). While the data presented in this paper are phenomenological, and do not point directly to pathogen influences, we use pathogens as an example in developing a framework that integrates the expected phylogenetic component of FIG. 1. Change in phylodiversity with size class (seedlings, density dependence that we have outlined with a 0–50 cm tall; saplings, 50 cm tall to 1 cm dbh; poles, 1–5 cm mechanistic, causal hypothesis. Invertebrate herbivores, dbh; trees, .10 cm dbh) for 28 plots (36 m2 for seedlings to poles; 0.16 ha for trees, hence separation by dashed vertical with a species richness comparable to pathogens, might line); box indicates median (heavy line) and quartiles, whiskers interact with plant phylodiversity in a similar way reach to points 1.5 times the interquartile range. Units of (Weiblen et al. 2006). both APd and NTPd are one standard deviation of a ran- Within a plant community, a plant pathogen species dom distribution of phylogenetic distance (Pd), zeroed to can have a single species of host plant (‘‘monophagy’’) or the distribution’s mean. (a) Average phylodiversity (APd ¼ NRI), which becomes more positive with decreasing mean several (‘‘polyphagy’’). Monophagous pathogen–host relatedness among all pairs of taxa in the sample. (b) Nearest dynamics have been well explored and tend to result in taxon phylodiversity (NTPd ¼ –NTI, which is the mean of the classic negative density-dependent effects in the plant relatedness to the most closely related taxon to each taxon). (Burdon and Chilvers 1982). This will be the case either Abbreviations are as follows: NRI, net relatedness index; NTI, nearest taxon index. in a monoculture of the plant species or in a mixture with nonhost plant species. However, the population dynamics that result when pathogens can attack several (Webb et al. 2002, Cavender-Bares et al. 2004) must be plant species in a community are more complicated, and plesiomorphic to a clade, whereas negative interactions have been addressed mainly in the context of apparent must occur among the most closely-related taxa within competition (Price et al. 1988, Alexander and Holt the clade. For instance, if disease cross-species suscep- 1998). Here, an increase in numbers of one plant species tibility is highest in sister taxa, while drought tolerance leads to an increase in pathogen inoculum, which could important for long-term survival on ridges is shared then affect other susceptible plant species in the among many taxa in a phylogenetic family, we should community. In a tropical rain forest, the hundreds of see a simultaneous increase in nearest taxon phylodiver- plant species present are exposed to thousands of sity (NTPd) and a decrease in ‘‘deeper,’’ average potential fungal pathogens, mostly unidentified (Arnold phylodiversity (APd). If only taxa with a particular et al. 2000, Gilbert et al. 2002). Some of the pathogens synapomorphy can survive in a particular place to are monophagous and some have many hosts (Lindblad July 2006 PHILODIVERSITY AND SEEDLING DYNAMICS S129

FIG. 2. Theoretical framework for the phylogenetic effects of pathogens. (a) Model of spread of single pathogen to other host plants (i þ 1ton) in the community. The output vector of probabilities of cross-infection is a function of the phylogenetic relatedness of other taxa to the host plant (i), the degree of polyphagy in the pathogen, and the ‘‘ease’’ of host switching to unrelated host plants. (b) Model of community-wide cross-infection by all pathogens. The output is a matrix of cross-infection probabilities, declining from a high rate (H) in closely related taxa to a low rate (L) in unrelated taxa. The mean baseline rate (l) depends on the mean ‘‘ease’’ of pathogen host switching to unrelated taxa, both historically, and with contemporary rapid evolution in ecotypes. Note that the lower half of the matrix can differ from the upper half if the probability of infection from plant i to j is not equal to the probability from j to i.

2000, Woolhouse et al. 2001, Gilbert et al. 2002). Mitter 1993, Farrell 2001), a pathogen that can infect Individual plant species might host dozens of species of one species can most easily become adapted to fungal symbionts (Frohlich and Hyde 1999, Arnold et phylogenetically closely related species. At the same al. 2001), many with negative effects on the host (reviews time, while phylogenetic conservatism of morphology in Gilbert 2002, 2005). Host susceptibility to pathogens and biochemistry is pervasive in nature, so is parallel will also be influenced in plastic ways by plant evolution, or homoplasy, of similar characters in abundance, phenological state, season, nutrient avail- unrelated lineages. Hence, pathogen adaptations to ability, and other factors which stress a plant. infection barriers might also be effective in phylogeneti- Analyzing and understanding this complicated net- cally unrelated plants, which partially explains why work of interactions and effects might be greatly some pathogens can infect many unrelated plants in a simplified by modeling pathogen host breadth as a community (Weste and Marks 1987, Eckenwalder and phylogenetic function (Webb et al. 2002; cf. Weiblen et Heath 2001). al. 2006). The barriers for a pathogen to infect a host In general, the probability that a single pathogen can plant are morphological and biochemical. Moreover, infect a particular set of hosts in a plant community because both morphology and plant secondary chem- should decline with increasing phylogenetic separation istry are often conserved phylogenetically (Farrell and among the hosts, but with some host-sharing across some S130 CAMPBELL O. WEBB ET AL. Ecology Special Issue apparently random plant species as well (Fig. 2a). Eco- systems, then the persistence and recovery of phyloge- logical association, through consistent community co- netic diversity might be assisted by nature itself. occurrence of unrelated but common species, might ACKNOWLEDGMENTS facilitate more distant host jumps. Considering the many pathogen species likely to be present, we expect variation C. O. Webb thanks David Ackerly for informative discussions about phylogenetic structure and David Peart for in (1) the number of hosts that pathogens can attack, (2) many discussions on the nature of density dependence. Karen the slope of their phylogenetically determined drop-off Garrett provided feedback in the development of the con- from closely related cohosts to less related nonhosts, and ceptual model. C. O. Webb was funded during analysis and (3) the extent of nonphylogenetic host ‘‘jumps.’’ Combin- writing by a grant from the National Science Foundation ing the variation in all these pathogens, we expect that the (DEB-0212873) and during the field work by a graduate fellowship (GER-9253849) and a Small Grants for Exploratory net interspecific cross-infection probability will decline Research (SGER) grant to David Peart (DEB-9520889). We with increasing phylogenetic distance, down to a low thank the various branches of the Indonesian government that baseline that represents the frequency of phylogenetically permitted the fieldwork, and Darmawan and Suryadi, C. O. unrelated host ‘‘jumping’’ (Fig. 2b, matrix element L). Webb’s field assistants at Gunung Palung. The review com- In this way, pathogens, which are already known to ments of Mark Westoby and an anonymous reviewer are much appreciated. exert strong influences on plant survival in natural systems, might operate in a phylodiversity-dependent LITERATURE CITED manner and might underlay the phylodiversity-depend- Alexander, H. M., and R. D. Holt. 1998. The interaction ent seedling survival observed at Gunung Palung. The between plant competition and disease. Perspectives in Plant most powerful way to confirm the phylogenetic-dis- Ecology Evolution and Systematics 1:206–220. tance-dependent signal in pathogen interactions will be APG [Angiosperm Phylogeny Group]. 2003. An update of the Angiosperm Phylogeny Group classification for the orders to experimentally cross-inoculate pathogens in repli- and families of flowering plants: APG II. 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SUPPLEMENT Supertree phylogeny ﬁles and taxonomic list (Ecological Archives E087-115-S1). Ecology, 87(7) Supplement, 2006, pp. S132–S149 Ó 2006 by the Ecological Society of America

PLANT DEFENSE SYNDROMES

1,3 2,4 ANURAG A. AGRAWAL AND MARK FISHBEIN 1Department of Ecology and Evolutionary Biology and Department of Entomology, Cornell University, Ithaca, New York 14853 USA 2Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi 39762 USA

Abstract. Given that a plant’s defensive strategy against herbivory is never likely to be a single trait, we develop the concept of plant defense syndromes, where association with specific ecological interactions can result in convergence on suites of covarying defensive traits. Defense syndromes can be studied within communities of diverse plant species as well as within clades of closely related species. In either case, theory predicts that plant defense traits can consistently covary across species, due to shared evolutionary ancestry or due to adaptive convergence. We examined potential defense syndromes in 24 species of milkweeds (Asclepias spp.) in a field experiment. Employing phylogenetically independent contrasts, we found few correlations between seven defensive traits, no bivariate trade-offs, and notable positive correlations between trichome density and latex production, and between C:N ratio and leaf toughness. We then used a hierarchical cluster analysis to produce a phenogram of defense trait similarity among the 24 species. This analysis revealed three distinct clusters of species. The defense syndromes of these species clusters are associated with either low nutritional quality or a balance of higher nutritional quality coupled with physical or chemical defenses. The phenogram based on defense traits was not congruent, however, with a molecular phylogeny of the group, suggesting convergence on defense syndromes. Finally, we examined the performance of monarch butterfly caterpillars on the 24 milkweed species in the field; monarch growth and survival did not differ on plants in the three syndromes, although multiple regression revealed that leaf trichomes and toughness significantly reduced caterpillar growth. The discovery of convergent plant defense syndromes can be used as a framework to ask questions about how abiotic environments, communities of herbivores, and biogeography are associated with particular defense strategies of plants. Key words: Asclepias; cardenolides; chemical ecology; cluster analysis; coevolution; Danaus plexippus; herbivory; latex; milkweed; monarch butterfly; phylogenetically independent contrasts; phytochemistry; plant–insect interactions.

INTRODUCTION role in the development of ideas on both the ecology and Understanding the macroevolution of adaptive traits evolution of plants and insect herbivores, two of the has inspired biologists for decades, yet has been most diverse lineages of eukaryotes (Ehrlich and Raven challenging to study (Schluter 2000). The difficulty lies 1964, Coley 1983, Farrell et al. 1991, Farrell and Mitter in (1) identifying the trait, or more commonly suites of 1993, 1998, Becerra 1997, Berenbaum 2001). In this traits, responsible for the adaptation of interest, (2) paper, we present a new synthesis of ideas on the having adequate phylogenies to examine macroevolu- macroevolution of plant defense traits, with an attempt tionary patterns, (3) distinguishing apparent adaptation to identify the relative roles of phylogenetic history and from ‘‘random’’ evolution along a diversifying phylog- ecological variables in shaping the expression of suites of eny, and (4) matching the origins of adaptations to defense traits within species. We then test some of our contemporaneous biotic and abiotic environmental proposed ideas and assumptions utilizing new data on factors that have likely driven adaptive changes. Despite the phylogeny and defense of milkweeds (Asclepias these difficulties, the study of plant–herbivore interac- spp.). tions has contributed substantially to understanding the Although it is convenient to consider plant defense as macroevolution of adaptive traits. Research on the a single trait, plants typically utilize a broad arsenal of macroevolution of plant defense has played a prominent defensive traits against herbivores (Duffey and Stout 1996, Romeo et al. 1996). Even when a plant species is Manuscript received 21 January 2005; revised 9 May 2005; apparently defended by a single type or class of defense accepted 21 June 2005; final version received 1 August 2005. chemical, there are typically many specific forms of Corresponding Editor (ad hoc): C. O. Webb. For reprints of those compounds (Berenbaum et al. 1986, Malcolm this Special Issue, see footnote 1, p. S1. 1991, Bennett and Wallsgrove 1994, Becerra 1997). 3 E-mail: [email protected] 4 Present address: Department of Biology, Portland State Thus, it is more useful to think about plant defense as a University, Portland, Oregon 97207 USA. suite of traits, which might include aspects of a plant’s S132 July 2006 PLANT DEFENSE SYNDROMES S133 nutritional quality (e.g., proteins and antiproteins), share a syndrome due to common ancestry, then physical characteristics (e.g., spines, trichomes, and leaf common ancestry is sufficient to explain the association, toughness), toxicity (e.g., cyanides and alkaloids), and adaptive advantage need not necessarily be invoked. phenology, regrowth capacity (i.e., tolerance), and Like trade-offs among supposedly redundant traits, the indirect defenses (e.g., volatiles and branching architec- correlated evolution of traits composing syndromes ture). Synergistic interactions between multiple traits is needs to be tested explicitly rather than assumed. particularly important in potentially providing a greater Although plant defense syndromes have received little level of defense than would be possible if the traits were attention in the past (but see Feeny 1976, Kursar and present independently (Broadway and Duffey 1988, Coley 2003), we suggest that where distantly related Gunasena et al. 1988, Berenbaum et al. 1991, Stapley plant species share a common assemblage of herbivores, 1998). they are likely to defend themselves with similar Nonetheless, most attention historically has consid- strategies. For example, plant species attacked primarily ered defenses as singleton strategies, with the typical by vertebrate grazers should employ quite different prediction that there should be trade-offs among differ- strategies (e.g., spines, leaf toughness, and out-of-reach ent antiherbivore strategies (because they could be costly morphologies) than plants primarily attacked by cater- and/or redundant) (Steward and Keeler 1988, Herms pillars (e.g., trichomes, toxins, and parasitoid-attracting and Mattson 1992). We argue that this reasoning, in its volatiles). Perhaps the original ‘‘plant defense syn- simplest form, is inaccurate, because plants do simulta- drome’’ was the set of traits proposed to be associated neously employ multiple defense traits. Of course, with highly apparent plants (Feeny 1976). Feeny argued particular plant defenses might trade off against each that apparent plants like oak trees were bound to be other, but this should not be the a priori expectation for found, and thus defended with suites of traits that make any two defense-related traits. If plant defenses, like them nutritionally poor: high concentrations of tannins, most adaptations, are composed of multiple traits, they low water and nitrogen content, and tough leaves. We might be organized into coadapted complexes (Dobz- agree that such patterns are likely to have evolved hansky 1970). repeatedly to converge on distinct syndromes of The categorization of an organism’s phenotype into defensive traits, even in distantly related plant taxa. So suites of potentially covarying traits has a long tradition why have most ecologists not explicitly considered plant in biology, including the concepts of guilds and defense syndromes? We believe that the traits of syndromes (Root 1967, Grime 1977, Fægri and van importance in defense against herbivores are too often der Pijl 1979, Simberloff and Dayan 1991, Chapin et al. unobservable by the naked eye of naturalists, and 1993, Cunningham et al. 1999, Fenster et al. 2004, therefore have escaped attention. Wilson et al. 2004). The syndrome concept has been rightly criticized as overly simplistic when applied Plant defense syndromes in communities uncritically (e.g., Waser et al. 1996); however, suites of versus taxonomic groups covarying traits can be usefully employed in some cases to infer adaptation to specific selective contexts. For The concept that plant species within a particular example, suites of traits associated with hummingbird- community may coexist due to trade-offs in fitness- pollinated flowers (i.e., red color, tubular morphology, enhancing traits is an old and central paradigm in plant extended anther position, dilute nectar concentration, ecology (reviewed by Tilman and Pacala [1993]). This etc.) have repeatedly evolved from those of bee- logic was later applied to plant defense traits for pollinated ancestors in diverse lineages (McDade 1992, coexisting species. Van der Meijden et al. (1988) Beardsley et al. 2003, Castellanos et al. 2003, 2004, proposed that suites of traits associated with resistance Fenster et al. 2004). Patterns of natural selection vs. regrowth (tolerance) might represent alternative imposed by birds and bees implicate these pollinators strategies for coexisting temperate herbaceous plants. as the agents favoring floral syndromes (e.g., Schemske Spurred by Janzen’s (1974) study of tropical blackwater and Bradshaw 1999). Although any two traits in a rivers and emerging physiological plant defense theories syndrome can be positively or negatively (or not at all) (Bryant et al. 1983), Coley and colleagues hypothesized correlated across taxa, the plant defense syndrome that plant species colonizing light gaps would have hypothesis rejects the prediction that any two plant divergent (trading-off) suites of defense traits, compared defenses are redundant, and thus should be negatively to species colonizing the more resource-poor understory associated across species (Steward and Keeler 1988, in tropical forests (Coley 1983, 1987, Coley et al. 1985). Twigg and Socha 1996, Rudgers et al. 2004). Defense Such trade-offs explained divergent patterns of herbi- syndromes as whole, however, can trade off if they truly vory in different microenvironments and also likely represent alternative adaptive strategies. contributed to the maintenance of diversity at larger If a syndrome has independently evolved multiple spatial scales. Recent experimental work on phylogeneti- times, it suggests that the selective forces driving the cally paired species from white sand and clay soil evolution of these convergent adaptations are common habitats of Peru support the hypothesis (Fine et al. 2004, and widespread. If, however, closely related species 2006). S134 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue

Kursar and Coley (2003) have also expanded these et al. 2004). Of course, such an association does not ideas to explicitly consider the evolution of convergent prove adaptation, but, at minimum, implies an evolu- defense syndromes in tropical trees. They argue that tionary association between traits and ecology. trees fall along an escape–defense continuum: extreme The macroevolution of syndromes has also been ‘‘escape’’ species are predicted to have few chemical studied within clades. Within the plant genus Penstemon, defenses, but rapid synchronous leaf expansion, and low species have repeatedly evolved suites of traits associated leaf nutritional quality during expansion; extreme with hummingbird pollination from Hymenoptera- ‘‘defense’’ species have high chemical defense, low pollinated ancestors (Thomson et al. 2000, Wilson et nutritional quality, and asynchronous leaf expansion al. 2004). The repeated evolution of particular plant (Kursar and Coley 2003). Using phylogenetically defense trait combinations within a taxonomic group independent contrasts, Silvertown and Dodd (1996) (e.g., genus) is suggestive of ‘‘syndromes in clades.’’ showed that herbaceous vs. woody plants had distinct Traits associated with induced resistance to herbivores types of chemical defenses (tannins vs. alkaloids), evolved multiple times in the cotton genus, Gosspium consistent with classic apparency theory (Feeny 1976). (Thaler and Karban 1997). Work with ant-associated In each of these examples, the general finding has been and nonant-associated Acacia spp. may reveal patterns that unrelated plant species within a particular com- of divergent defensive strategies in plants with altered munity have converged on a suite of similar strategies species interactions (Rehr et al. 1973, Seigler and that maximize fitness, given a particular set of ecological Ebinger 1987, Heil et al. 2000, 2002, 2004). Recent interactions. Thus within a regional community, plant work by Becerra and colleagues (Becerra 1997, Becerra species can converge on a few defense syndromes, yet the et al. 2001) demonstrates that there has been convergent divergent strategies (across the syndromes) also can evolution of terpene chemical defense and ‘‘squirt-gun’’ promote the coexistence of species. defense combinations in tropical Bursera spp. Species of The approach of examining syndromes within com- specialist Blepharida beetles typically consume plants munities is an ecological question, first and foremost, with similar defense traits, which are not necessarily the and begins with identifying the traits of species that most closely related species or those with geographically occur within a plant community irrespective of their similar ranges (Becerra 1997, Becerra and Venable evolutionary relationships. If patterns of defensive traits 1999). Thus, traits can covary within a clade and are consistently occur among coexisting taxa, then the predicted to be associated with particular interactions patterns of defense among species could be explained (although convergent taxa do not necessarily coexist). by either shared phylogenetic history or convergence due The approach of examining syndromes within a clade to similar selective agents causing repeated evolution of requires an accurate plant phylogeny and the existence a defensive syndrome. We propose that phylogenetic of two or more syndromes (suites of traits). Thus, if history should provide the null hypothesis to explain the suites of traits converge in different parts of the distribution of defense traits among species (Silvertown phylogeny, the question becomes this: Are there and Dodd 1996). The alternative hypothesis is that consistent ecological correlates that match these differ- species’ defensive traits are evolutionarily labile, and ent syndromes? A key difference from the community species have repeatedly evolved homoplasious (i.e., approach is that syndromes within a closely related set independent, but similar) solutions to biotic and abiotic of species in a clade can surface in geographically environmental challenges. For instance, in a temperate separated species that do not naturally coexist. The grassland community, patterns of plant defense could community approach specifically starts with species that consistently fall into a few categories (e.g., toxic and ant- coexist under relatively similar ecological conditions. defended vs. tolerant and poor nutritional quality), but Although similar in trying to disentangle the effects of each of these categories might be phylogenetically phylogenetic history and ecology, the ‘‘community’’ and homologous (e.g., evolving once each in legumes and ‘‘clade’’ approaches start at the opposing ends of the grasses). Alternatively, unrelated taxa could converge on phylogeny–ecology continuum. homoplasious suites of traits in response to the same or similar selective forces (Fine et al. 2004). Defense syndromes in milkweeds (Asclepias) In community studies of syndromes, phylogenetic There are ;130 species of Asclepias native to North information need not be extremely detailed, and America, including Mesoamerica and the Caribbean taxonomic information might be sufficient. If two (Woodson 1954; M. Fishbein et al., unpublished manu- species within a genus have different suites of defense script). Approximately six additional species are native traits and are ecologically divergent (i.e., have different to temperate South America (Bollwinkel 1969). The types of species interactions, live in different micro- situation is more complicated in Africa, where up to 200 habitats, etc), then selection is a reasonable hypothesis species have been or could potentially be included in the for the differences between the close relatives; if multi- genus Asclepias, depending on the breadth of circum- ple, phylogenetically independent congeneric pairs show scription and phylogenetic relationships among African similar suites of defensive traits and divergent ecologies, and American species (Fishbein 1996). However, pre- convergence can be invoked (Conway-Morris 2003, Fine liminary analyses suggest that American and African July 2006 PLANT DEFENSE SYNDROMES S135

TABLE 1. Species sampled for defense-related traits, effects on herbivore performance, and phylogenetic relationships.

Species Native Range Asclepias amplexicaulis Sm. Eastern USA Asclepias asperula (Decne.) Woodson ssp. asperula Southwestern USA and Mexico Asclepias californica Greene Western USA Asclepias cordifolia (Benth.) Jeps. Western USA Asclepias curassavica L. Neotropics Asclepias eriocarpa Benth. Western USA Asclepias exaltata L. Eastern USA Asclepias fascicularis Decne. Western USA and Mexico Asclepias hallii A. Gray Western USA Asclepias hirtella (Pennell) Woodson Central USA Asclepias incarnata L. ssp. incarnata Eastern USA Asclepias incarnata L. ssp. pulchra (Ehrh. ex Willd.) Woodson Eastern USA Asclepias oenotheroides Schltdl. & Cham. Southwestern USA, Mexico, and Central America Asclepias perennis Walter Southeastern USA Asclepias purpurascens L. Eastern USA Asclepias speciosa Torr. Western USA Asclepias sullivantii Engelm. ex A. Gray Central USA Asclepias syriaca L. Eastern USA and Canada Asclepias tuberosa L. Eastern USA, southwestern USA, and Mexico Asclepias variegata L. Eastern USA Asclepias verticillata L. Eastern USA and Canada Asclepias viridis Walter Eastern USA Gomphocarpus cancellatus (Burm. f.) Bruyns Southern Africa Gomphocarpus fruticosus (L.) W.T. Aiton Africa

species belong to distinct sister clades (Rapini et al. 2003; of the guilds. Some of the herbivores, such as Monarchs M. Fishbein et al., unpublished manuscript), and thus (Danaus plexippus) and Milkweed aphids (Aphis nerii), Asclepias may be considered an exclusively American are broadly distributed and feed on many milkweeds. genus of ;150 species. In this study, we employ the Other groups, such as the cerambycid beetles in the results of the most intensively sampled study of genus Tetraopes have radiated with the milkweed genus, Asclepias to date (M. Fishbein et al., unpublished and each of the 24 Tetraopes species is primarily manuscript), focusing on the American species as a associated with a single milkweed species (Farrell and framework for investigating macroevolution of defense Mitter 1998). Thus, each milkweed species is likely traits. Of the species we studied, all are perennial, some attacked by several herbivores, although the species are clonal, and genets are probably very long-lived composition and guilds attacking particular species vary (Table 1). In addition, most of the species are relatively widely. An important future goal in the study of plant rare in the landscape; those that are common now, such defense macroevolution is to detail the particular insect as A. syriaca in eastern North America, were probably communities that correspondingly attack plants with much less abundant and widespread prior to the clearing different defense syndromes. of the eastern deciduous forest by colonists. The community of insects on milkweed thrives by Plants in the genus Asclepias have been of major either actively avoiding defenses in the plant or by importance in the development of theories about plant– consuming, sequestering, and advertising these same herbivore and tri-trophic interactions, and historically defenses. Probably the two most potent aspects of the focus has been primarily on cardenolides as a plant herbivore resistance in milkweeds are the production of defense and the Monarch butterfly as the herbivore cardenolides and latex. Cardenolides are bitter tasting (Fig. 1) (Brower et al. 1967, 1972, Malcolm et al. 1989). steroids that occur in all milkweed tissues, including the However, the herbivore community of Asclepias spp. latex, that act by disrupting the sodium and potassium consists of tens of species that are principally host- flux in cells, and have toxic effects on most animals specific insects (Weiss and Dickerson 1921, Rothschild (Malcolm 1991). The sticky white latex is delivered via et al. 1970, Wilbur 1976, Blakley and Dingle 1978, Price specialized canals (laticifers) to most plant parts, and and Willson 1979, Morse 1985, Malcolm 1991, Fordyce can be copiously exuded upon tissue damage (Fig. 1). and Malcolm 2000, Agrawal and Malcolm 2002). These Latex of milkweed has been strongly implicated as a herbivores fill almost every conceivable feeding guild: physical impediment to herbivory (Dussourd and Eisner aphids feed on the phloem, beetles and caterpillars chew 1987, Zalucki et al. 2001). Other potentially defensive the leaves, flies mine the leaves, hemipterans eat the and nutritional constituents that could influence herbi- seeds, weevils bore through the stem and eat the pith, vores include leaf toughness, trichome density, water and beetle larvae bore through the roots. content, nitrogen content, and specific leaf area (Matt- For example, the common milkweed, A. syriaca, has son 1980, Coley 1983, Haddad and Hicks 2000, Lavoie 12 reported insect herbivores, including members of all and Oberhauser 2004). Most of these traits show plastic S136 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue

FIG. 1. (A) Common milkweed (Asclepias syriaca) viewed from above early in the growing season. (B) A newly hatched monarch caterpillar (Danaus plexippus). Before it can get its first meal, the caterpillar must graze the trichomes and avoid the droplets of latex. Exposure to latex is reduced by clipping laticifers in a circle and feeding on the tissue in the middle. variation within species, are genetically variable in accounting for plant mortality, our common garden had natural populations of single species, and are highly approximately 12 replicate plants per species (mean 6 SE, variable across species (Table 2). 11.8 6 0.7). All measurements were taken from newly Using 24 species of Asclepias, we begin to address the expanded, undamaged leaves of plants in the common role of convergent evolution in giving rise to antiherbi- garden. vore defense syndromes. We specifically addressed the We measured cardenolide concentrations as digitoxin following questions: (1) What are the pairwise associa- equivalents (grams per gram dry tissue) extracted from tions of defense traits across taxa? Are defense traits 50 mg dry leaf tissue (n ¼ 5 replicates/species); we typically negatively correlated as predicted by redun- employed a spectrophotometric assay modified from dancy or trade-off theory, are they positively correlated Nelson (1993). We adapted the assay for sampling using as predicted by syndrome theory, or are they not a microplate reader (PowerWave X, Bio-Tek Instru- correlated at all? (2) Do species cluster into syndromes ments, Winooski, Vermont, USA). Field-collected leaf of common patterns of defense trait expression? (3) Are tissue was kept on ice, then frozen, freeze-dried, ground phylogenetic relationships among Asclepias species with a mortar and pestle, and weighed in 2-mL boil- inferred from DNA sequences congruent with patterns proof tubes. To each tube, we added 1.9 mL of 95% of defense trait similarity? If so, we can conclude that ethanol, tubes were then vortexed, and floated in a phylogenetic history is sufficient to explain plant defense sonicating water bath (658C) for 10 min. We then trait associations; if not, there is a suggestion for centrifuged the tubes at 5000 rpm for five minutes at convergence in defense syndromes. And (4) Are partic- room temperature. Two 45-lL aliquots of the super- ular defense traits or syndromes associated with the natant from each tube were then pipetted into the wells performance of a common oligophagous herbivore of of a 96-well plate, one above the other (active sample milkweeds (monarch caterpillars, Danaus plexippus)? and blank, respectively). Each plate also contained six samples of digitoxin for the standard curve used to MATERIALS AND METHODS determine concentrations of cardenolides (0.125–3 mg/ Measuring plant traits mL; Sigma Chemical, St. Louis, Missouri, USA). We then added 90 lL of ethanol to the blanks and 90 lLof Seeds for 24 species of milkweeds (Table 1) were 0.15% 2,204,40-tetranitrodiphenyl (TNDP) in ethanol to obtained from field collections, nurseries, and native plant the active samples. Finally, 70 lL of 0.1 mol/L aqueous seed suppliers. We germinated seeds by nicking the tips NaOH was added to all wells to make the solutions basic and placing the seeds on moist filter paper. Seedlings were and to catalyze the colorimetric reaction. After 15 grown in potting soil (;10-cm pots) in growth chambers minutes, all wells in the plate were read at 620 nm using for one month and out-planted in a completely random- the microplate reader. ized common garden in a plowed field at the Koffler We measured latex from 5–10 replicates from each Scientific Reserve at Jokers Hill, Southern Ontario (448030 species by cutting the tip off (0.5 cm) an intact leaf in the N, 798290 W; information available online).5 After field and collecting the exuding latex onto a 1 cm disc of filter paper. Latex stopped flowing after ;10 s, all latex 5 hhttp://www.zoo.utoronto.ca/jokershilli was absorbed on the filter paper, and this disc was placed July 2006 PLANT DEFENSE SYNDROMES S137

TABLE 2. Seven putative defensive traits of milkweeds. Ranges for the actual trait values for treatment and for family and species means are presented, followed by percentage variation (in parentheses).

Putative defense trait Plastic variation Full-sib variation Species level variation Latex (mg) 1.05–1.35 (29%) 0.9–4.5 (400%) 0–4.8 (infinite) Cardenolides (% dry mass) 2.7–4.0 (48%) 1.7–3.9 (129%) 2–16 (800%) 2 Trichomes (no. hairs/cm ) NA 580–1090 (88%) 0–2019 (infinite) Toughness (g) NA 96–128 (33%) 58–177 (205%) C:N 7.8–9.0 (15%) 11–14 (27%) 10–15 (50%) Water content (%) NA NA 78–90 (15%) 2 Specific leaf area (mm /mg) NA NA 8–23 (188%) Notes: In experiments, we have characterized the level of variation in these traits among control Asclepias syriaca plants and those experimentally damaged by herbivores (plastic variation) (Van Zandt and Agrawal 2004a; A. A. Agrawal, unpublished data), 23 full-sibling families of A. syriaca (Agrawal 2004b, 2005), and 24 different species of Asclepias (Agrawal 2004a, this study). All data were collected from at least five individuals (per treatment, family, or species) typically growing in growth chambers (plastic variation) or independent field common gardens (full-sibling and species variation). There were significant differences between treatments, families, and species for all traits measured (ANOVA for all traits, P 0.05); families and species vary continuously between the extremes presented. on top of another dry filter paper disc in a 24-well plate. contrasts (PICs) (Felsenstein 1985). We calculated PICs The discs were dried at 608C and then weighed to the using the maximum-likelihood (ML) estimate of the microgram. This method has proven to be highly re- phylogeny of the 24 species of Asclepias (Fig. 2) for peatable (Van Zandt and Agrawal 2004a, Agrawal 2005). which defense traits were measured (Table 2), based on We assessed the trichome density of 5–10 replicate noncoding plastid DNA (cpDNA) sequences (rpl16 plants from each species by counting the tops and intron and trnC–rpoB intergenic spacer; M. Fishbein et bottoms of leaf discs (28 mm2) under a dissection al., unpublished manuscript). We obtained sequences microscope. Leaf discs were taken from the tips of directly, using standard protocols, and manually aligned leaves. Water content was assessed by first weighing leaf them (M. Fishbein et al., unpublished manuscript). The discs wet and again after drying in an oven (608C). best fitting nucleotide substitution model under the ML Specific leaf area (SLA) was calculated as the area of the criterion (general time reversible plus invariant sites plus leaf disc divided by the dry mass. This measure can be gamma [GTRþIþC]) was selected using hierarchical thought of as an indicator of thickness or density; leaves likelihood ratio tests and ML trees were sought using with a higher SLA are typically thin and have greater a standard heuristic search strategy (using levels of herbivory (Scha¨ dler et al. 2003). PAUP*4.0b10; Swofford 2001) (see Fishbein et al. We measured leaf toughness on 10 replicate plants 2001). Clade support was assessed with Bayesian from each species with a force gauge penetrometer (Type probabilities estimated under the GTRþIþC model, 516; Chatillon, Largo, Florida, USA) that measures the assuming uninformative priors for model parameters mass (in grams) needed to penetrate a surface. We and tree topologies (using MrBayes 3.0b4, Ronquist and sandwiched the leaf between two pieces of Plexiglas, Huelsenbeck 2003) (see Fishbein and Soltis 2004). each with a 0.5-cm hole, pushed the probe of the Markov chains were iterated for 5 3 105 generations penetrometer through the leaf, and recorded the with trees sampled during the first 105 generations maximum force required for penetration. For each leaf, discarded as transitory samples. The ML tree for these we measured toughness on each side of the mid-rib; 24 species did not differ significantly from relationships these two measures were averaged and used as a single found in a comprehensive analysis of 105 species of data point per plant. Asclepias (M. Fishbein et al., unpublished manuscript; Total leaf carbon (C) and nitrogen (N) concentration results not shown). was measured from five replicates from each species by We calculated PICs using Felsenstein’s (1985) meth- microcombustion, using 5 mg of dried ground leaf od, as implemented in COMPARE (available online).6 material in an Elemental Combustion System 4010, We have no a priori evidence concerning rates of CHNS-O analyzer (Costech Analytical Technologies, evolution of defense traits. Thus, we conducted PIC Valencia, California, USA). We used the C:N ratio as analyses under the assumptions of (1) equal branch indicator of plant nutritional quality. lengths, corresponding to a speciational model of Means and standard errors of the seven defense- evolution (Mooers et al. 1999), and (2) branch lengths related traits for each of the 24 milkweed species are estimated from cpDNA sequences. However, the branch reported in Appendix A. lengths estimated from molecular data caused patho- Statistical analyses of phylogenetic and logical behavior in the PIC analysis: several exceedingly nonphylogenetic trait associations short internodes (Fig. 2) resulted in widely inflated Pairwise correlations among traits were calculated using raw trait values and phylogenetically independent 6 hhttp://compare.bio.indiana.edui S138 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue

FIG. 2. Maximum-likelihood phylogram of the 24 species of Asclepias employed in this study. Numbers indicate Bayesian posterior probabilities. Letters to the right of taxon names represent defense syndrome clusters (see Fig. 4 and Results: Assessing defense syndromes). estimates of contrasts involving these branches. Thus, (cf. Housworth and Martins 2001). Starting with a tree the few data points involving these nodes drove the in which all clades with posterior probabilities ,0.70 in pattern of correlation among contrasts (results not Fig. 2 were collapsed, we generated 100 trees that shown), which often differed dramatically from those randomly resolved all polytomies using MacClade 4.06 found in equal branch length analyses. Despite the (Maddison and Maddison 2003). We present 95% drawbacks of assuming equal probabilities of change conﬁdence limits for our correlation coefﬁcients by across all branches of the phylogeny, this seems excluding the lowest and highest 2.5% of the r values. preferable to alternative assumptions about unknown We conducted PIC analyses, as described, with COM- rates of evolution. Thus, we only present the results PARE on each of these 100 trees. based on assuming equal branch lengths. We used a hierarchical cluster analysis to produce a The nodes of the phylogeny subtended by extremely defense phenogram (cf. Becerra’s ‘‘chemograms’’) (Be- short branches correspond to areas of uncertainty in the cerra 1997, Legendre and Legendre 1998) to group phylogeny of Asclepias (see posterior clade probabilities species by expression of defense traits. We used the mean in Fig. 2). A consequence of this uncertainty is possible values for each of the seven plant traits measured for error in the PIC analysis, due to using an incorrect each species to generate a phenogram. Mean trait values phylogenetic estimate. To examine this source of error, were transformed to Z scores (mean ¼ 0, SD ¼ 1) so that we conducted PIC analyses on alternative topologies they were measured on a comparable scale. Following and assessed the robustness of PIC-based correlations Becerra’s (1997) model, we employed Ward’s method for July 2006 PLANT DEFENSE SYNDROMES S139

TABLE 3. Pairwise correlations of defense-related traits among Asclepias species.

Water Latex Trichomes Toughness content Cardenolides SLA (mg) (no. hairs/cm2) (g) C:N (%) (% dry mass) (cm2/mg) Latex (mg) 0.60** 0.10 0.04 0.21 0.13 0.59** Trichomes 0.62** 0.10 0.17 0.04 0.05 0.43* (no. hairs/cm2) (0.47–0.70) Toughness (g) 0.06 0.05 0.32 0.19 0.06 0.26 (0.25–0.10) (0.27–0.18) C:N 0.03 0.18 0.43* 0.25 0.25 0.26 (0.25–0.10) (0.43 to 0.115) (0.18–0.455) Water content 0.29 0.01 0.34 0.17 0.27 0.39 (%) (0.32 to 0.045) (0.08–0.165) (0.35 to 0.13) (0.30 to 0.01) Cardenolides 0.08 0.1 0.01 0.24 0.16 0.15 (% dry mass) (0.16–0.02) (0.015–0.195) (0.095–0.105) (0.32 to 0.14) (0.065–0.265) SLA (cm2/mg) 0.57** 0.45* 0.15 0.07 0.57** 0.21 (0.58 to 0.42) (0.495 to 0.295) (0.24–0.055) (0.215–0.01) (0.46–0.61) (0.12–0.27) Notes: Correlation coefficients (r) above the diagonal are uncorrected for bias due to phylogenetic history; those below the diagonal are phylogenetically independent contrasts (Felsenstein 1985) assuming a speciational model of evolution (with the 95% confidence limits, based on random resolution of poorly supported nodes, shown in parentheses). SLA (last column) is specific leaf area. None of the significant correlations represents a trade-off in defenses (see Results). * P , 0.05, **P , 0.01; these values are also highlighted in boldface type; df ¼ 22. P 0.1; df ¼ 22. linkage and Euclidean distances, which combines sub- that is statistically independent of phylogenetic relation- groups (initially building from one species) at each ships. Stochastically evolving traits will generate clusters iteration so as to minimize the within-cluster ANOVA of taxa statistically indistinguishable from the estimated sum of squares (Wilkinson 1997). Alternate joining phylogeny. Significant deviations in the phenotypic algorithms provided qualitatively similar relationships. clustering could be caused by convergent adaptation of Clusters were designated by distances from nodes; nodes traits or other processes that generate trait correlations separated by a distance of ,0.5 were included in the that are independent of association due to phylogenetic same cluster, whereas nodes separated by .0.5 were history. This test is similar in aim to tests of trait placed in separate clusters (Wilkinson 1997). We further conservatism (e.g., Ackerly and Donoghue 1998, Cav- examined the strength to which particular traits ender-Bares et al. 2004). An advantage to our approach contributed to differences among clusters using discrim- is the ability to consider the associations among all traits inant function analysis (Wilkinson 1997). simultaneously, with the concomitant disadvantage that To investigate whether factors related to geographical the conservatism of a single trait cannot be assessed. distribution were associated with the repeated emergence of trait combinations, we conducted a preliminary test Effects of defense traits on caterpillars for an association between geography and membership To assess how individual plant traits and defense in the hierarchical clusters (Thaler and Karban 1997, syndromes affect the performance of one natural feeder Becerra and Venable 1999). Biogeographical associations of milkweeds, we conducted a bioassay with monarch of defense clusters were investigated by contingency table caterpillars (Danaus plexippus) (Fig. 1). We placed single analysis. The bulk of species we studied have ranges in eggs of freshly hatched caterpillars on all plants (n ’ 12 eastern North American (United States and Canada, east for each of the 24 plant species) in the field. Caterpillars of the Tall Grass Prairie–Great Plains ecotone) or were from a laboratory colony established from local western North American (west of the ecotone) (Table 1). individuals collected the previous summer and main- Congruence between the ML phylogeny of Asclepias tained on frozen milkweed foliage. Each caterpillar was and hierarchical clustering of species based on defense caged in a spun polyester bag (Rockingham Opportu- traits was assessed using the test of Shimodaira and nities, Reidsville, North Carolina, USA) on the apical Hasegawa (SH test) (Shimodaira and Hasegawa 1999, meristem with four fully expanded leaves. After five Goldman et al. 2000), as implemented in PAUP* 4.0b10 days, we recorded mortality and collected and weighed (Swofford 2001), using RELL resampling to estimate each living caterpillar. site likelihoods. Statistical incongruence between the To assess whether plant species that were in the three phylogeny of Asclepias and clustering of species by different defense clusters (see Results) showed differ- defense traits would indicate that the evolution of ential resistance to monarch caterpillars, we conducted a defensive traits does not passively track phylogeny, but one-way ANOVA. The species cluster was used as the instead is evolutionarily labile, and suggests phylogeneti- factor (each species was assigned to one cluster) and cally independent derivations of associations among mean monarch mass and percentage survival on each traits. This approach evaluates whether associations species were the response variables (total n ¼ 24). In among traits generate hierarchical similarity among taxa addition, we employed multiple regression to assess the S140 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue

FIG. 3. Signiﬁcant pairwise correlation between raw values of latex production and trichome density among 24 Asclepias species (see Table 2). Raw correlations and those of phylogenetically independent contrasts (PIC) (Felsenstein 1985) are signiﬁcant and of similar magnitude.

effects of plant traits on monarch performance across between SLA and water content and between leaf C:N the 24 species. For this analysis, we used species means ratio and toughness were stronger and statistically for each of the seven traits (cardenolides, latex, significant compared to the raw correlations. In trichomes, water content, specific leaf area, toughness, addition, a weak and nonsignificant negative correlation and C:N ratio) and mean caterpillar mass and percent- between leaf water content and toughness had a nearly age mortality as the response variables. We employed a significant phylogenetically independent correlation stepwise multiple regression with backwards removal (P (P ¼ 0.10; Table 3). ¼ 0.15 to enter or remove) (Wilkinson 1997). Correlations based on PICs were generally robust to phylogenetic uncertainty (Table 3). For the latex– RESULTS trichome, latex–SLA and SLA–percentage water corre- Pairwise correlations between traits lations, every one of the randomly resolved topologies generated significant correlations. The trichome–SLA Three of the 21 raw pairwise correlations (and five correlation was significant in just 41% of the random based on phylogenetically independent contrasts, PICs) resolutions, although the upper bound of the 95% among seven defense-related traits were statistically confidence limit (r ¼0.295) was still strongly negative. significant (Table 3). This observed frequency of The C:N–toughness correlation was less robust, with phylogenetically independent correlations is unlikely to only 12% of the random resolutions remaining signifi- have occurred by chance (binomial expansion test, P ¼ cant. The only instance of a nonsignificant PIC 0.0028) (Zar 1996). Each of the significant raw correlation that was found to be significant in randomly correlations involved just three traits showing comple- resolved topologies was the C:N–trichome correlation, mentary patterns of variation. Species with lower but this occurred in only 3% of the resolutions. Overall, specific leaf areas (SLA) exhibited higher leaf trichome little bias can be attributed to using the ML phylogeny densities and latex production (Fig. 3). Species with in the face of weak support for a number of clades. lower SLA also exhibited lower water content (P ¼ 0.06), as expected (SLA is derived from leaf dry mass). Recall Assessing defense syndromes that SLA is a physiological plant trait, often indicative Our hierarchical cluster analysis of the seven defense- of rapid growth and high palatability to herbivores, and related traits revealed three distinct clusters (Fig. 4). For that low SLA might defend against herbivory. Leaves of illustrative purposes only, in post hoc analyses, we species with high C:N ratios were also tougher, although determined that clusters differed strongly from one this relationship was not significant (P ¼ 0.13). Thus, we another (MANOVA results from discriminant function found no indications of trade-offs among defense traits. analysis: Wilks’ lambda ¼ 0.033, F14,30 ¼ 9.688, P , Indeed, all correlations were ‘‘positive’’; the significant 0.001; see Appendix B). The analysis also indicates how negative correlations in Table 2 represent negative each trait correlated with each discriminant function associations between nutritional quality and defense. (Table 4); although most traits contributed substantially The phylogenetically independent correlations did not to at least one function, cardenolides and leaf toughness differ substantially from raw correlations (Table 3, contributed the least to both. We also identified which Fig. 3). The phylogenetically independent correlation traits were significantly heterogeneous among the three July 2006 PLANT DEFENSE SYNDROMES S141

FIG. 4. A defense phenogram that depicts similarity among 24 species of Asclepias generated by hierarchical cluster analysis of seven defense-related traits. Tightly clustered species are defensively similar and can be considered to form defense syndromes, A–C (see Results: Assessing defense syndromes).

clusters (Table 4). These analyses revealed that most or B, respectively. Clusters A and C are strategies that traits were expressed differently in the three defense both represent the coupling of a trait increasing the clusters, and again cardenolides and leaf toughness were reward to herbivores (nitrogen or SLA) with high the least distinct. defense allocation. The three defense clusters were not The three clusters (Fig. 4, Table 4) represent species significantly associated with distribution in eastern vs. with combinations of: (A) high nitrogen (i.e., low C:N) western North America (v2 ¼ 8.77, df ¼ 5, P ¼ 0.12). coupled with high physical defense traits (trichomes, latex), (B) very high C:N ratio, coupled with tough Phylogenetic congruence test leaves and low water content (hard to eat, little reward), The phylogenetic relationships suggested by the and (C) low C:N and SLA coupled with high defense trait cluster analysis (Fig. 4) are significantly cardenolides. Although differences in cardenolides were incongruent with relationships estimated from non- not significantly different between clusters (Table 3), we coding cpDNA sequences (Fig. 2). The difference in found 17% and 50% higher levels in cluster C than in A log likelihood [ln(L)] between the maximum likelihood

TABLE 4. The relationship between seven defensive traits and defensive clustering of milkweed species. Coefficients of standardized canonical discriminant functions (CDFs) indicate how each trait contributes to the two factors generated by discriminant analysis (Wilkinson 1997). Trait values (mean 6 SE) appear for the three defense syndromes (clusters) identified in this study. Significance of differences among traits tested by ANOVA.

CDF1 CDF2 Cluster A (n ¼ 4) Cluster B (n ¼ 6) Cluster C (n ¼ 14) P Latex (mg) –0.495 0.404 3.13 6 0.72 1.28 6 0.56 0.31 6 0.09 ,0.001 Trichomes (no. hairs/cm2) –0.907 0.316 1630.08 6 146.57 355.40 6 185.22 337.47 6 67.11 ,0.001 Toughness (g) –0.089 0.285 111.08 6 17.93 131.00 6 12.01 104.86 6 5.53 0.136 C:N 0.637 0.703 11.48 6 0.08 14.09 6 0.32 11.66 6 0.21 ,0.001 Water content (%) 0.102 –0.388 0.83 6 0.01 0.81 6 0.01 0.84 6 0.01 0.088 Cardenolides (% dry mass) –0.011 –0.008 4.97 6 0.67 3.89 6 0.76 5.85 6 1.11 0.508 Specific leaf area (mm2/mg) –0.456 –0.284 9.98 6 0.69 10.41 6 0.99 15.80 6 1.02 0.002 S142 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue

FIG. 5. Schematic depiction of the lack of congruence between the molecular phylogeny of Asclepias and the defense trait phenogram (see Fig. 4). tree for the sequence data (–ln(L) ¼ 4225.61) and the survival is difficult to explain and is discussed below (see highest likelihood tree congruent with the phenogram the Discussion). (–ln(L) ¼ 4532.10) was 306.49, which was highly DISCUSSION significant (SH test: P , 0.05). Thus, multivariate defense clusters did not come about only as a conse- The concept of convergent expression of suites of quence of tracking the speciational history of Asclepias traits acting as syndromes has been a useful framework (Fig. 5). for conceptualizing adaptations in ecological communities. For example, plant ecologists have proposed that Effects of defense traits on caterpillars specific suites of traits might be associated with particular types of environmental stress (Grime 1977, When plant species were classified by defense cluster Chapin et al. 1993). In animal ecology, the guild concept (Fig. 4), we found no difference in the performance of is used to characterize species that exploit the same type monarch caterpillars among groups (mass, F ¼ 2.460, 2,20 of resources in a similar manner (Root 1967). Species P F P ¼ 0.111; survival, 2,21 ¼ 2.080, ¼ 0.150). Nonethe- forming a guild can share a common set of traits, but are less, in multiple regression analyses, plant traits signifi- explicitly not necessarily unified by phylogenetic related- cantly explained monarch performance (Fig. 6) (overall ness. Nonetheless, in the past there was limited ability to 2 model for mass, R ¼ 0.33, F2,20 ¼ 4.906, P ¼ 0.018, disentangle the role of phylogenetic history and con- trichome coefficient ¼0.130, P ¼ 0.022, toughness vergence in generating such species groups. For exam- coefficient ¼0.698, P ¼ 0.041; overall model for ple, in the plant pollination literature, phylogenetic 2 survival, R ¼ 0.26, F1,22 ¼ 7.566, P ¼ 0.012, latex analyses of convergence on plant traits associated with coefficient ¼ 0.060, P ¼ 0.012). The latter positive bee and hummingbird pollination have only recently correlation between latex and percentage of caterpillar been employed (McDade 1992, Fenster et al. 2004, July 2006 PLANT DEFENSE SYNDROMES S143

Wilson et al. 2004). Other assessments of syndromes have also recently adopted a phylogenetically explicit approach (Cunningham et al. 1999, Fine et al. 2004). Thus, we see our concept of plant defense syndromes as an extension and phylogenetic modernization of classic plant defense theory (e.g., Feeny 1976).

Correlations between traits In our study of Asclepias, none of the measured defense-related traits showed the bivariate trade-off predicted by traditional redundancy theory. Indeed, all of the pairwise correlations between defensive traits were positive; the significant correlations with a negative sign were between nutritional quality and defense, thus, not indicating a trade-off among traits providing resistance. Using phylogenetically independent contrasts among cotton (Gossypium) species, Rudgers et al. (2004) also found no trade-off between the production of extrafloral nectaries (EFNs) and trichomes, or between EFNs and chemical defense glands; however, they did find a negative association between trichomes and glands. This negative correlation could reflect allocation costs of the traits or redundancy in their ecological functions. Given that the costs and ecological functions of most defense- related traits are unknown, however, in this paper we have argued that any two traits should not be a priori predicted to negatively covary. This same conclusion has been reached in a recent meta-analysis of intraspecific genetic correlations of defense traits (Koricheva et al. 2004).

We found a positive (phylogenetically independent) FIG. 6. Effects of milkweed defensive traits on monarch correlation between plant production of latex and caterpillar growth. Seven plant traits were assayed, and only trichomes (Fig. 3). In an analysis of the correlation the significant predictors are shown: (A) trichome density and between the same two traits across 23 genetic families of (B) leaf toughness. Residual growth refers to the residuals from the statistical model with the other factor in the analysis. A. syriaca, we found no correlation (Agrawal 2005). Although other intraspecific correlations are needed, Armbruster et al. (2004) and others have argued that such correlations across species, but not within species, our short-term assays this protective effect is, thus, are suggestive of adaptation over constraint. We perhaps an artifact. speculate that this association could be due to the fact A notable outlier in the positive correlation between that latex is a water-intensive defense, and the protection trichomes and latex (Fig. 3) was A. sullivantii, which has from water loss by trichomes facilitates use of latex. high latex production but no trichomes. Two other Additionally, latex has widely been reported to function possible independent origins of species with high latex as a defense against monarch caterpillars and other production coupled with the absence of trichomes are A. milkweed herbivores (Dussourd and Eisner 1987, humistrata (southeastern USA) and a small clade of Zalucki et al. 2001, Agrawal and Van Zandt 2003, Mexican species (A. glaucescens, A. elata, A. mirifica, Agrawal 2004b, 2005), suggesting that the two strategies and A. lynchiana), neither of which were sampled in the might be employed in concert to produce a synergistic current study. These species (including A. sullivantii) all defense. Although we did not detect a negative effect of have pronounced depositions of epicuticular wax (Fish- latex on monarchs in the current study, it is perhaps in bein 1996), which we speculate may be a substitute for this situation where plants derive benefits from employing dual strategies. We speculate that the positive trichomes in a physical defense syndrome. correlation we observed between latex production and Thus, in summary, the admittedly adaptationist percentage caterpillar survival is an artifact of a hypothesis under the defense syndrome concept is not correlation between latex and some unmeasured trait. of specific a priori trade-offs, but a prediction that the It is also possible that plant species with higher levels of multivariate trait complex is grouped such that costs are latex prevented caterpillars from feeding, which pro- minimized and defense is maximized, given the biotic and tected them from death due to other plant defenses; in abiotic environment of the species. Defense syndromes S144 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue

FIG. 7. The plant defense syndrome triangle. Low nutritional defense syndrome is consistent with that outlined for apparent plants by Feeny (1976); a similar group was found in this study. Tolerance follows the fast-growth and high-edibility pattern outlined by Coley (Coley et al. 1985, Kursar and Coley 2003). Nutrition and defense is a strategy that couples a toxic defense or barrier to feeding with relatively high edibility and digestibility. SLA denotes speciﬁc leaf area. themselves might trade-off if they represent multivariate quality (from the herbivore’s perspective) and high strategies targeted at alternative ecological interactions. expression of defense traits. In cluster A, low C:N ratio (i.e., relatively high nitrogen levels) is coupled with high Defense syndromes in milkweeds and beyond latex production and trichome density. In cluster C, We distinguished three syndromes (clusters) in our both a low C:N ratio and high speciﬁc leaf area (SLA; multivariate analysis of seven defense-related traits, i.e., relatively concentrated plant tissue) is coupled with which grouped as plant species employing (A) high higher levels of cardenolides than the other clusters. In physical defenses, (B) low nutritional quality, and (C) the case of the cluster-A species, repeated evolution of a putatively high chemical defense (Figs. 1 and 3). We latex–trichome associated syndrome is statistically sup- found that, overall, our clustering of plant species by ported (Table 3, Fig. 3) defense traits was not congruent with the molecular Although this analysis of Asclepias focused primarily phylogeny of the group. At a minimum, this means that on resistance, plant growth traits and tolerance are likely the defense characteristics of Ascelpias are evolutionarily to be an important component of particular defense labile, and do not simply track phylogenetic history. For syndromes (van der Meijden et al. 1988, Fornoni et al. example, in the well-resolved group of three species that 2004). For example, Kursar and Coley (2003) made the co-occur in eastern North America (A. syriaca, A. argument that a trade-off between defense and growth tuberosa, and A. exaltata) all three clusters are promoted divergent strategies among tropical tree represented. In addition, it is apparent that some taxa, species. We offer the ‘‘defense syndrome triangle’’ although distantly related, have converged on similar hypothesis to include all defense categories (Fig. 7). defense phenotypes. Presumably other traits, such as plant traits that affect Although we have not reconstructed the relative herbivore preference and attraction or facilitation of timing of changes in plant nutritional quality and enemies of herbivores will also be important to integrate expression of defense traits, we assume that when such into the concept of plant defense syndromes. traits are associated, changes in defense traits follow Nearest to the origin of the two axes of edibility/ after divergences in nutrition traits rather than the digestibility and toxicity/barriers is the low nutritional reverse. For two of the three defense clusters (A and C in quality syndrome (Fig. 7). This syndrome, empirically Fig. 4) there is an association between high resource demonstrated in Asclepias (Syndrome B in Fig. 4, Table July 2006 PLANT DEFENSE SYNDROMES S145

4), very closely mirrors Feeny’s (1976) grouping of limiting the time of divergence among species and defensive traits for apparent plants, and Coley et al.’s permitting denser taxon sampling. (1985, Kursar and Coley 2003) ‘‘defense syndrome’’ for It is important to bear in mind that our working plants that have evolved in low-resource environments. hypothesis for the phylogeny of Asclepias (M. Fishbein Note that the defense syndrome concept does not et al., unpublished manuscript) is not highly resolved with implicate a particular selective agent in generating the strong statistical support (see Fig. 2). The lack of suite of adaptations. Feeny hypothesized that the resolution reflects the reality of the apparently rapid selective agents were herbivores under the constraint of initial radiation of Asclepias. It is also important to the probability of being found; Coley also hypothesized consider the effect of our assumption of speciational that herbivores were the selective agent; however, this evolution of morphology (i.e., equal morphological was presumed to be constrained by the abiotic (resource) branch lengths across the tree) in calculating phyloge- environment. The tolerance or escape syndrome (pro- netically independent contrasts (PICs). Both of these posed by Kursar and Coley [2003], and not investigated factors could bias our estimates of evolutionary in the current study) predicts a lack of resistance traits in correlations among traits. We found that estimated very fast-growing and nutritive plants. Finally, our correlations among PICs were very robust to uncer- study revealed two syndromes with intermediate levels tainty regarding the correct relationships in poorly of edibility coupled with barriers to consumption supported regions of the tree. Conversely, the assump- (Syndromes A and C, Fig. 4, Table 4). In some ways, tion concerning the mode of evolution of defense traits this mixed strategy is similar to Feeny’s prediction for had a dramatic effect on the sign and magnitude of unapparent plants (i.e., qualitative defenses that cannot correlations. Ideally, an individual model of evolution be overcome by generalist herbivores, but specialists should be developed for each trait in order to accurately feed on these plants with impunity). The important estimate correlations (e.g., Pagel 1999, Lewis 2001). distinction is the implicit recognition that these toxins or Because of the relatively small number of taxa (i.e., low barriers to consumption (latex, trichomes, cardenolides, sample size) and uncertainty regarding the correct etc), typically have a dose-dependent (or quantitative) phylogenetic relationships, we did not pursue individu- effect on specialist herbivores (Berenbaum et al. 1989, alized fitting of evolutionary models for the defense Adler et al. 1995, Agrawal and Kurashige 2003, Agrawal traits. We did attempt to analyze correlations assuming and Van Zandt 2003, Agrawal 2004a, b, 2005). Plants that rates of morphological evolution were accurately with high levels of toxicity or barriers to feeding, estimated by branch lengths inferred from the rates of however, are not predicted to have particularly high or nucleotide substitution in the cpDNA sequences used to low levels of nutritional quality (Fig. 7). We reach this estimate the tree. Correlations estimated under this conclusion based on the reasoning that plants with assumption differed substantially from those reported extreme investments in toxins should not have the ability here. to produce very nutritive tissues; likewise plants with Although we are not comfortable assuming equal very low nutritive quality should not need to invest in branch lengths across the tree (which is almost certainly toxins. incorrect), we prefer this assumption to the use of molecular branch lengths. Generally, there is little Caveats evidence for correspondence between rates of molecular The performance of monarch butterfly caterpillars on and morphological evolution (Bromham et al. 2002). the 24 milkweed species did not differ on plants from the Specifically, the extreme branch length heterogeneity for three syndromes. This suggests that plant species might the molecular data is likely unreasonable for the have alternate mechanisms to achieve similar levels of morphological traits under study here. Species of defense, and these might be driven by environmental Asclepias that exhibit very low levels of divergence in differences in the habitats in which these species live cpDNA sequences can be dramatically different in naturally. Alternatively, our analysis should be viewed morphology, whereas species that differ only subtly (A. as preliminary and is limited by (1) our somewhat fascicularis, A. verticillata, A. perennis, and A. incarnata) arbitrary distinction of clusters (see Materials and are separated by branches that are orders of magnitude methods: Effects of defense traits on caterpillars), (2) longer than those separating morphologically diverse unmeasured defense traits (epicuticular waxes, pro- species (see Fig. 2). Given the apparently poor teases, etc.), (3) a limited sample of species, and (4) correspondence between rates of molecular and mor- limited information on the ecology of each species that phological evolution, we prefer the assumption of would be informative for why particular defense speciational changes in morphology over any assump- syndromes were expressed across these species. Thus, tion about specific rates across lineages. further analyses are needed. In addition, our approach was to sample broadly across Asclepias, but more Directions detailed analyses of well-resolved clades would also be We see the identification of syndromes as a starting valuable. Such fine-scale sampling would better control point to test alternative hypotheses for why plant for the confounding effects of unmeasured traits by defenses have converged. In this regard, the biotic and S146 ANURAG A. AGRAWAL AND MARK FISHBEIN Ecology Special Issue abiotic environment could conspire to drive particular and field; Robie Mason-Gamer and Steve Lynch for contribu- syndromes. For example, plant trichomes are known to tions to the phylogenetic study of Asclepias; David Goyder, Mark Chase, and Marlin Bowles for providing DNA samples; have many functions, including defense against herbi- Bobby Gendron for seeds; Steve Malcolm for discussions on vory as well as a barrier against evapotranspirative cardenolide analysis; and Paul Fine, Marc Johnson, Rick water loss (e.g., Woodman and Fernandes 1991, Karban, Marc Lajeunesse, Peter Price, Jennifer and Jon Thaler, Agrawal and Spiller 2004). Thus, trichomes might be and Cam Webb for comments and discussion. 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APPENDIX A Means and standard errors for seven defense-related traits of the 24 species of milkweed employed in this study (Ecological Archives E087-116-A1).

APPENDIX B The three defense clusters separated in multivariate space (Ecological Archives E087-116-A2). Ecology, 87(7) Supplement, 2006, pp. S150–S162 Ó 2006 by the Ecological Society of America

THE GROWTH–DEFENSE TRADE-OFF AND HABITAT SPECIALIZATION BY PLANTS IN AMAZONIAN FORESTS

1,2,3,9 3 4 5 6 PAUL V. A. FINE, ZACHARIAH J. MILLER, ITALO MESONES, SEBASTIAN IRAZUZTA, HEIDI M. APPEL, 7 8 6 1 M. HENRY H. STEVENS, ILARI SA¨ A¨ KSJA¨ RVI, JACK C. SCHULTZ, AND PHYLLIS D. COLEY 1Department of Biology, University of Utah, Salt Lake City, Utah 84112 USA 2Environmental and Conservation Programs and Department of Botany, Field Museum of Natural History, Chicago, Illinois 60605 USA 3Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109-1048 USA 4Department of Forestry, Universidad Nacional de la Amazonı´a Peruana, Plaza Serafı´n Filomeno 246, Iquitos, Peru 5Department of Biology, McMaster University, Hamilton, Ontario L8S4K1 Canada 6Pesticide Research Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802 USA 7Department of Botany, Miami University, Oxford, Ohio 45056 USA 8Zoological Museum, Centre for Biodiversity, FIN-20014, University of Turku, Finland

Abstract. Tropical forests include a diversity of habitats, which has led to specialization in plants. Near Iquitos, in the Peruvian Amazon, nutrient-rich clay forests surround nutrient-poor white-sand forests, each harboring a unique composition of habitat specialist trees. We tested the hypothesis that the combination of impoverished soils and herbivory creates strong natural selection for plant defenses in white-sand forest, while rapid growth is favored in clay forests. Recently, we reported evidence from a reciprocal-transplant experiment that manipulated the presence of herbivores and involved 20 species from six genera, including phylogenetically independent pairs of closely related white-sand and clay specialists. When protected from herbivores, clay specialists exhibited faster growth rates than white-sand specialists in both habitats. But, when unprotected, white-sand specialists outperformed clay specialists in white- sand habitat, and clay specialists outperformed white-sand specialists in clay habitat. Here we test further the hypothesis that the growth–defense trade-off contributes to habitat specialization by comparing patterns of growth, herbivory, and defensive traits in these same six genera of white-sand and clay specialists. While the probability of herbivore attack did not differ between the two habitats, an artificial defoliation experiment showed that the impact of herbivory on plant mortality was significantly greater in white-sand forests. We quantified the amount of terpenes, phenolics, leaf toughness, and available foliar protein for the plants in the experiment. Different genera invested in different defensive strategies, and we found strong evidence for phylogenetic constraint in defense type. Overall, however, we found significantly higher total defense investment for white-sand specialists, relative to their clay specialist congeners. Furthermore, herbivore resistance consistently exhibited a significant trade-off against growth rate in each of the six phylogenetically independent species-pairs. These results confirm theoretical predictions that a trade-off exists between growth rate and defense investment, causing white-sand and clay specialists to evolve divergent strategies. We propose that the growth–defense trade-off is universal and provides an important mechanism by which herbivores govern plant distribution patterns across resource gradients. Key words: Amazon; ecological gradient; growth–defense trade-off; habitat specialization; herbivory; phenolics; phylogenetic control; rainforest; reciprocal-transplant experiment; terpenes; tropical trees.

INTRODUCTION ment and out-compete other plants that are not so The regional diversity of plant species arises, in part, closely suited to the local conditions (Ashton 1969, because a given species is restricted to a subset of Cody 1978, Bunce et al. 1979). However, herbivore– environmental conditions. But how and why does this plant interactions can also contribute to the evolution of habitat specialization occur? The most common expla- habitat specialization. Theoretical work has demonstra- nation is that habitat specialists are physiologically ted that herbivores can alter competitive relationships adapted to growing in their particular abiotic environ- among plants, especially when there is spatial heterogeneity of resources (Louda et al. 1990, Grover and Holt 1998). Empirical studies at the population and com- Manuscript received 8 February 2005; revised 29 April 2005; accepted 3 May 2005; ﬁnal version received 11 July 2005. munity levels have documented that herbivores can Corresponding Editor: A. A. Agrawal. For reprints of this reduce plants’ potential distributions, restricting them to Special Issue, see footnote 1, p. S1. a subset of the habitats that they might physiologically 9 Present address: Department of Ecology and Evolu- tionary Biology, University of Michigan, Ann Arbor, tolerate (Parker and Root 1981, Louda 1982, 1983, Michigan 48109-1048 USA. E-mail: paulﬁ[email protected] Louda and Rodman 1996, Olff and Ritchie 1998, S150 July 2006 PLANT TRADE-OFFS AND SPECIALIZATION S151

Carson and Root 2000, Harley 2003). Thus, herbivores cally plastic as opposed to genetically controlled can play a major role in determining which species of adaptations to a particular habitat. plants dominate in a community, as well as in which Thus, to test whether the growth–defense trade-off habitats a species will be successful. contributes to habitat specialization in white-sand and The lowland Amazonian rainforest near Iquitos, Peru clay forests, we combined field observations and a provides an ideal system to study habitat specialization reciprocal-transplant experiment to ask the following and the role of herbivores. Forests in the Iquitos area questions: (1) Are there differences in herbivore abun- grow on a mosaic of soil types; including red clay soils dance in the two habitats? (2) Is there a difference in the and extremely infertile white-sand soils (Kauffmann et impact of herbivory in the two habitats, suggesting al. 1998). The two soil types lie immediately adjacent to selection for greater defense investment in white-sand each other, the boundaries are well defined, and each soil habitats? (3) Do white-sand and clay specialists differ in type is associated with a distinctive flora (Gentry 1986, their type of defensive strategy or in their amount of Va´ squez 1997, Fine 2004). White-sand forests are much defense investment? Are these differences phylogeneti- more resource limited than clay soil forests (Medina and cally constrained or repeatedly and independently Cuevas 1989, Coomes and Grubb 1998, Moran et al. evolved? (4) Are defensive traits in white-sand and clay 2000). Resource availability theory proposes that specialists affected by resource-driven phenotypic plas- resource-limited species will have slower growth rates ticity? (5) Do white-sand and clay specialists follow the predictions of the growth–defense trade-off? and higher optimal levels of defense, reflecting the decreased ability of a resource-limited plant to compen- MATERIALS AND METHODS sate for tissues lost due to herbivory (Janzen 1974, Coley Study site and study species et al. 1985, Coley 1987b). Thus we predict that species growing in white-sand forests should evolve to allocate We conducted this research in the Allpahuayo- relatively more resources to defense than species Mishana National Reserve near Iquitos, Peru (38570 S, 0 growing in clay forests (Fine et al. 2004). 73824 W). This 57 600-ha reserve is at ;130 m elevation Recently, we reported the results of a reciprocal- and receives more than 3000 mm of precipitation during transplant experiment of 20 species of seedlings from six the year, with no distinct dry season (Marengo 1998). genera of phylogenetically independent pairs of white- Many white-sand specialist trees belong to the same sand and clay specialist plants (Fine et al. 2004). We genera as neighboring clay forest specialists, allowing for manipulated the presence of herbivores and found that a phylogenetically controlled experiment using edaphic clay specialists grew significantly faster than did white- specialist species. For a reciprocal-transplant experi- sand specialists in both habitats when protected from ment, we chose 20 common white-sand and clay herbivores. But when herbivores were not excluded, specialists from six genera from five families (see Fine et al. [2004] for a phylogeny). The genera were Mabea white-sand specialists out-performed clay specialists in (Euphorbiaceae), Oxandra (Annonaceae), Pachira (Mal- white-sand forests, and clay specialists grew faster than vaceae sensu lato), Parkia (Fabaceae), Protium (Burser- white-sand specialists in clay forests. These results aceae), and Swartzia (Fabaceae). Each genus was strongly supported the existence of a growth–defense represented by one white-sand specialist and one clay trade-off, with habitat specialization being enforced by specialist, except for Protium, which was represented by herbivores (Fine et al. 2004). six clay specialists and four white-sand specialists. Here, we test further the predictions of the growth– Designation of habitat for each species was accom- defense trade-off by comparing species-level patterns of plished by extensive inventories (Fine 2004, Fine et al. growth, herbivory, and defense in this same phylogeneti- 2005) as well as consultation of local floras and other cally diverse group of tree species. We predicted that published species lists from the western Amazon closely related species specialized to contrasting soil (Va´ squez 1997, Ruokolainen and Tuomisto 1998, types should diverge in traits that confer defense vs. Jørgensen and Le´ on-Ya´ nez 1999, Garcı´ a et al. 2003). those that confer growth. We investigated the evidence for such differential investment while controlling for Nitrogen availability phylogeny. Therefore, any differences in defense alloca- To test for differences in nitrogen availability between tion found between closely related white-sand and clay white-sand and clay habitats, we filled 27 nylon stocking specialists can be inferred to be traits derived for habitat bags filled with 8 g of Rexyn 300 (H-OH) analytical specialization. This phylogenetically controlled ap- grade resin beads. In May 2002, we placed the ion- proach enabled us to investigate the degree of constraint exchange resin bags beneath the litter layer and root mat involved in the type and amount of defense, and to at the organic material–mineral soil interface at our separate this from the repeated and independent white-sand and clay sites (Binkley and Matson 1983). evolution of defensive traits due to selection from The bags were collected after five weeks, extracted with similar ecological conditions. Second, examining defense KCl, and measured by standard techniques with an investment with a reciprocal-transplant experiment autoanalyzer (University of Wisconsin Soils Labora- allowed us to identify which traits (if any) are phenotypi- tory). Nitrate, ammonium, and root mat depth differ- S152 PAUL V. A. FINE ET AL. Ecology Special Issue ences were tested for significance between soil types with common in both white sand and clay forests: Protium a Wilcoxon signed-ranks test. (Burseraceae), Hevea and Mabea (Euphorbiaceae), Pachira (Malvaceae s.l.), and Macrolobium (Fabaceae). The reciprocal-transplant experiment In September 2000, in the same white-sand and clay sites We used a reciprocal-transplant experiment to test wherethewaspswerecollected,wesampled355 whether white-sand and clay specialists had different individuals in the field from .20 species of Protium, growth rates and defense investments as predicted by the two species of Hevea, two species of Mabea, two species growth–defense trade-off hypothesis. In addition, the of Pachira, and three species of Macrolobium. Most of reciprocal-transplant experiment allowed us to test for these species were found in only one of the two habitats. phenotypic plasticity of defense investments under Plants were 1–3 m tall (juvenile trees). We marked newly different edaphic and herbivore treatments. expanding leaves (or leaves that had already expanded In May 2001, we built 22 control and 22 herbivore but were not toughened) with small colored wires, from exclosures (3 3 3 3 2 m); half were located in clay forest 1–10 leaves or leaflets per plant. After five to seven and half in white-sand forest. We transplanted 880 weeks we estimated the amount of leaf area missing from seedlings from the six genera into the controls and the marked leaflet (0–100%). Average amount of leaf exclosures (see Fine et al. 2004). Using the results of the area missing was divided by number of days between reciprocal-transplant experiment reported in Fine et al. marking and the census (damage per day). These data (2004), we compared the amount of leaf and height were arcsine square-root transformed to improve growth of the plants grown in herbivore exclosures to normality, and a mixed-model ANOVA (including the the unprotected controls, and estimated the effect random factor genus and the fixed factor habitat) was herbivory had on growth rates for each white-sand performed on the data to test for differences in and clay specialist. This measure is referred to through- herbivory rate between white-sand and clay habitats. out as ‘‘protection effect.’’ The experiment lasted until February 2003 (21 mo after transplanting, 18 mo after Impact of herbivory (defoliation experiment) first data collection), at which point leaves were collected In February 2003, after collecting leaf material for to measure defensive traits. chemical analyses from all of the seedlings in the reciprocal-transplant experiment, we removed 100% of Insect abundance and species richness the remaining leaves to test the effect of defoliation on To evaluate differences in insect abundance and white-sand and clay specialists in the two habitats. After composition across habitats, we used a portable black three months, we counted the number of seedlings that light attached to a battery to attract insects in five white- survived defoliation. To compare mortality rates, we sand and five clay sites. During 8–20 December 2002, on averaged mortality for white-sand specialists and clay rain-free evenings between 1900–2000, the black-light specialists in each of the 44 controls and exclosures was illuminated and suspended above white sheets. We (Protium species in each control and exclosure were collected all insects from the families/orders Blattoideae, weighted to give each genus equal importance in the Coleoptera, Hemiptera, Homoptera, and Orthoptera. analyses). A fixed-factor ANOVA including the terms We excluded all obvious predators and collected all habitat (white-sand or clay), origin (white-sand or clay herbivorous insects from these five groups and counted specialist), and the origin 3 habitat interaction was used and identified them to order and family and then to assess the effects of origin and habitat on mortality separated them into morphospecies. Parasitoid wasps due to defoliation. Post hoc tests on the individual group were collected with malaise traps over a two-year period means were performed using the studentized t distribu- in 15 white-sand and nonwhite-sand forest sites in the tion (appropriate for equal sample sizes; Sokal and Allpahuayo-Mishana National Reserve (from 15 of the Rohlf 1995). same sites described in Fine [2004]) as a part of a much larger study on ichneumonid wasps (for detailed Defensive characteristics of white-sand methods see Sa¨ a¨ ksja¨ rvi [2003]). Since these parasitoid and clay specialists wasps attack either herbivorous insects (or predators of Comparing differences in herbivory and growth is the herbivorous insects), we would expect parasitoid diver- best method of comparing defense investment in white- sity and abundance to track herbivorous insect diversity sand and clay specialists, since this approach takes into and abundance in white-sand and clay forests. To test account the entire arsenal of plant defenses as experi- for differences between white-sand and clay habitats enced by the actual herbivores (cf. Simms and Rausher (both the black light trap data and the wasp data), 1987). However, to investigate which particular defen- Wilcoxon signed-ranks tests were conducted on the sive traits are deterring herbivores, we measured two ranked abundances and numbers of species. classes of chemical defenses, a physical defense, and the nutritional quality of white-sand and clay specialists. Field herbivory After the transplant experiment was completed, we For herbivory comparisons in addition to those from collected leaves from all surviving plants to compare the transplant experiment, we chose six genera that were defense investment in white-sand and clay specialists, July 2006 PLANT TRADE-OFFS AND SPECIALIZATION S153 and to assess the effect of habitat and treatment on the our total phenolics obtained as described with available plasticity of defense investment for each species. We foliar protein data to create a phenolic : protein ratio collected marked mature leaves that were produced after (Nichols-Orians 1991). plants were transplanted. We measured terpenes, total phenolics, toughness, and available protein for all Leaf toughness seedlings in the reciprocal-transplant experiment. Ter- A ‘‘penetrometer’’ (Chatillon Universal Tension and penes and phenolics are carbon-based secondary com- Compression Tester, Largo, Florida, USA) was used to pounds common to many families of plants, including puncture holes through the leaf (or leaflet) lamina to those in our research (Mabry and Gill 1979, Bernays et give a measure of toughness. It was impossible to test the al. 1989, Schultes and Raffauf 1990). Although phenolics pair of species from the genus Parkia, since both have and terpenes have alternative functions, they commonly bipinnately compound leaves, with leaflets not much function in herbivore deterrence (Mabry and Gill 1979, larger than the 3 mm diameter of the testing machine’s Bernays et al. 1989, Herms and Mattson 1992, Langen- rod. We standardized the punch position to midway heim 1994; but see Harborne 1991, Close and McArthur between leaf tip and base, between the midrib and the 2002). Increased toughness of leaves (sclerophylly) is a leaf margin, avoiding the main veins where possible. The mechanical antiherbivore defense that is commonly punch test measures a combination of shear and found worldwide in plants that live in resource-limited compressive strength and resistance to crack propaga- environments (Coley 1983, 1987a, Grubb 1986, Turner tion. For these reasons, it has been criticized as not 1994). Finally, available foliar protein is a good measure specifically measuring leaf toughness (Choong et al. of a plant’s nutritional quality. Moran and Hamilton 1992). Nevertheless, it is easy to perform in the field and (1980) hypothesized that plant nutrition can be consid- highly correlated with more specific tests to measure the ered a defensive trait if it can be selected for by herbivore physical properties of leaf toughness (shearing and attack. This can result if herbivores detect nutritional tearing parameters) (Edwards et al. 2000). differences and prefer plants with higher nutrition (cf. Scheirs et al. 2003). A second mechanism is if slow Soluble protein assays growth by herbivores due to low nutrition results in The amount of available foliar protein was measured higher predation rates (cf. Denno et al. 2002). at the Appel/Schultz laboratory using the same dried- leaf samples collected for the phenolics analyses. (See Chemical defenses Appendix A for detailed methods.) To compare terpene investment among the species, ;500 mg (fresh mass) leaves from the experimental Statistical analyses of growth and defensive traits seedlings were collected at the experimental sites in 2-mL Clay and white-sand specialists in each of the six glass vials and filled with dichloromethane (DCM). This genera were the experimental unit. Because there were leaf–DCM mixture was used for qualitative and four white-sand specialists and six clay specialists from quantitative analyses with gas chromatography (GC) the genus Protium, the responses for all Protium and gas chromatography–mass spectrometry (GCMS). individuals were weighted to give each genus equal (See Appendix A for detailed methods of terpene importance in the analysis. The four white-sand special- extraction and analysis.) ist Protium species were weighted at 0.25, the six clay For comparisons of total phenolics, ;2 g fresh mass specialist Protium species were weighted at 0.167, and of mature leaves of 16 individuals (8 protected and 8 species from all other genera were weighted at 1. We unprotected) from each species in the reciprocal-trans- used fixed factor ANOVAs to test for genus, origin (the plant experiment were collected and immediately placed difference between white-sand specialists and clay in plastic tubes containing silica gel desiccant. Leaves specialists), habitat (whether species responded differ- were later analyzed for phenolic compounds in the ently depending on where they were planted), and Appel/Schultz laboratory at Penn State University. treatment (whether defense investment differed depend- Whenever possible, bulk tannins were prepared to ing on whether the plants were exposed to herbivores). provide standards for the phenolic assays of individual Since we had a priori knowledge that different genera samples. This is a crude purification, and although would have different defensive strategies (i.e., some nonphenolic materials are unlikely to be present (Hager- species have terpene investment, others do not), we used man and Klucher 1986; H. M. Appel and J. C. Schultz, fixed-factor ANOVAs for defensive traits (genus was unpublished data), the product is merely a more treated as a fixed factor), since our ability to generalize representative sample of extractable polyphenols found our results in these analyses to unsampled genera is in the actual plant than is a commercial standard from limited. Subsequent to the overall test, individual group some other source. (See Appendix A for detailed means were compared with Tukey hsd post hoc tests. information on all methodology of phenolic extraction, purification, and analysis.) Because total phenolics likely Defense index function as an antiherbivore defense by precipitating Because different species of plants are likely to employ available protein (Herms and Mattson 1992), we divided different defensive strategies, we therefore devised a S154 PAUL V. A. FINE ET AL. Ecology Special Issue simple method to combine all measures of chemical plots to test our predictions that (1) growth and defense, leaf toughness, and available protein to inves- herbivory would be positively correlated, (2) growth tigate whether, for each genus, white-sand specialists and defense would be negatively correlated, and (3) were more defended than clay specialists. Values for herbivory and defense investment would be negatively phenolics, terpenes, and leaf toughness were averaged correlated. Hypotheses about the correlations of traits across both habitats and Z-transformed to give the were tested by the difference scores of the slopes and defense traits among the six pairs of white-sand and clay were evaluated for significance with one-tailed Wilcoxon specialists a mean of zero and a standard deviation of paired sign-rank tests (Zar 1999). one. Missing data was scored as zero. For available RESULTS protein, we standardized the inverse of the species averages, because a larger amount of available protein Differences in nutrient availabilities corresponds to lower defense. All four standardized Clay forest sites contained significantly more available defense variables were then summed to create a defense nitrogen (Z ¼ 3.53, P , 0.0004) than white-sand forests, index (DI). For each genus, the DI for the clay specialist more than twice as much available ammonium (Z ¼ was subtracted from the DI of the white-sand specialist. 2.71, P , 0.0061), more than an order of magnitude This method has the assumption that each of these four more available nitrate (Z ¼ 3.59, P , 0.0003), and a measures has equal weight, which is undoubtedly much thinner root mat (Z ¼ 4.89, P , 0.0001; Table 1). incorrect, but preferable than subjectively assigning different weights to defense types. These difference Habitat differences in herbivore abundance scores (DIWS –DICL) were used to test the prediction We found no significant differences in herbivore that white-sand specialists are more defended than clay abundance or species richness between habitats for all specialists with a one-tailed Wilcoxon paired signed- herbivores or any of the six orders of herbivorous insects ranks test (Zar 1999). that we collected (P . 0.05, Wilcoxon signed-ranks tests; Table 1). Of the 311 morphospecies collected, 208 Phylogenetic independence of growth, herbivory, were collected only once (67%). Of the morphospecies and defense traits collected more than once, 41 were collected only in In order to evaluate whether growth, herbivory, and white-sand forest, 28 were collected only in clay forest, defense traits were more similar in closely related genera, and 34 were collected in both forests (33% of the we mapped each of the indices listed above, as well as morphospecies collected more than once). For para- each individual defensive trait onto a phylogeny sitoid wasps, no statistical differences in abundance or representing the relationships among the six genera morphospecies diversity were found between white-sand and 20 species (see Fine et al. 2004). Using the program and nonwhite-sand forest sites (Table 1). Moreover, in Phylogenetic Independence 2.0, we tested whether traits the reciprocal-transplant experiment, mean effect of exhibited significant phylogenetic independence by protection for white-sand and clay specialists did not comparing the average contrast values (C-stat) among change between habitats (Fig. 1a, b). the actual trait values for the plant species to the distribution of contrast values created by randomly Differences in the magnitude of herbivore attack placing the trait values at the tips within the topology Clay specialists showed an average increase in growth 2000 times and testing for serial independence (TFSI) of 0.25 cm2/d in leaf area (paired t test, df¼5, t¼2.91, P (Abouheif 1999). If a trait is significantly phylogeneti- , 0.05) and 0.0018 cm/d in height (paired t test, df¼5, t¼ cally constrained, then the average C-stat for the actual 2.59, P , 0.05) when protected from herbivores, while value will be greater than 95% of the average contrast white-sand specialists grew just as well or better in the values generated by the randomization. unprotected vs. protected treatments. When the effect of herbivore protection on leaf area and height growth are Correlations of growth, defense, and herbivory data Z-transformed and summed, all genera show the same for the six genera pattern that clay specialists received a greater benefit from Species averages for growth (leaf area and height, herbivore protection than did white-sand specialists. averaged across habitats), the effect of herbivore During our study of field herbivory rates in the two protection on growth (arithmetic difference between habitats, plants in clay forest sites suffered more than the average leaf area and height with and without twice the herbivory rates on their new leaves than did protection, for each white-sand and clay genus averaged plants in white-sand sites (mixed model ANOVA, F1, 349 across habitats) and defenses, as described, were Z- ¼ 6.69, P , 0.01). Clay plants lost almost 23% of their transformed and analyzed by a method analogous to new leaves per month, while white-sand plants lost phylogenetically independent contrasts (Harvey and slightly .10% (Table 1). Pagel 1991). To test for trade-offs, we plotted the values for the six species pairs and analyzed the six slopes, to Habitat differences in the impact of herbivory see if the relationship between traits was consistent when As predicted, seedlings overall suffered higher mortal- controlling for phylogenetic relationship. We used these ity due to total defoliation in white-sand habitat than July 2006 PLANT TRADE-OFFS AND SPECIALIZATION S155

TABLE 1. Comparisons of white-sand and clay forests for leaf litter depth, nitrogen availability, young-leaf herbivory, and insect abundance and morphospecies richness (means 6 SE reported).

Variable Clay forest sites White-sand forest sites Root mat (cm) (N ¼ 44 plots) 0.91 6 1.0a 8.48 6 0.6 b Nitrogen availability (ppm) from ion-exchange resin bags (N ¼ 27 resin bags) – b a NO3 349.2 6 25.7 25.6 6 13.8 þ b a NH4 135.2 6 32.7 62.1 6 17.5 Total nitrogen 484.4 6 43.0 b 87.7 6 23.0 a Herbivory (% leaf eaten/mo) (N ¼ 355 individuals) 22.8 6 4.3 b 10.3 6 3.3 a Insect herbivore abundance ((no. individuals)(light trap)1h1) Total insect herbivore abundance 87.2 6 12.6 a 74.8 6 18.1 a Blattoid abundance 3.0 6 0.7 a 2.6 6 0.9 a Coleopteran abundance 20.0 6 9.0 a 22.4 6 7.8 a Hemipteran abundance 7.6 6 4.9 a 13.4 6 11.7 a Homopteran abundance 20.0 6 4.5 a 17.0 6 1.9 a Orthopteran abundance 36.6 6 8.2 a 19.2 6 3.6 a Insect herbivore species richness ((no. morphospecies)(light trap)1h1) Total insect herbivore morphospecies 45.0 6 4.3 a 34.8 6 3.9 a Blattoid morphospecies 2.6 6 0.5 a 2.4 6 0.8 a Coleopteran morphospecies 7.6 6 2.1 a 8.0 6 2.0 a Hemipteran morphospecies 3.2 6 1.0 a 2.0 6 0.4 a Homopteran morphospecies 15.8 6 2.8 a 11.4 6 2.0 a Orthopteran morphospecies 15.8 6 1.7 a 10.8 6 2.1 a Parasitoid wasp ((no. individuals)site1(malaise trap)1 for 2 yr) Total parasitoid wasp abundance 67.7 6 28.5 a 59.9 6 10.8a Total parasitoid species and morphospecies 25.5 6 6.4 a 22.0 6 3.3 a Note: Signiﬁcant differences between forests are indicated by different superscript letters within a row (mixed-model ANOVA, effect of habitat for herbivory, Wilcoxon signed-ranks tests between habitats for litter depth, nitrogen availability, insect abundance, and species richness).

they did in clay habitat (effect of habitat, F1,84 ¼4.96, P , the white-sand congener than in the clay congener, and 0.05). In addition, white-sand specialists suffered signifi- that our prediction of higher defense in the white-sand is cantly more mortality than did clay specialists in both supported (one-tailed Wilcoxon paired signed-ranks habitats (effect of origin, F1,84 ¼22.8, P , 0.0001; Fig. 2). test, T0.05(1), 6 ¼ 1, P , 0.05, Fig. 3). For phenolic compounds, white-sand specialists over- Differences in defense investment all had significantly higher values for both total Type of defense.—We found strong evidence for phenolics (effect of origin, F1, 292 ¼ 50.3, P , 0.0001) phylogenetic constraint for type of defense. The main and phenolic : protein ratios (F1, 292 ¼ 128.2, P , effect of genus was always significant for differences in 0.0001) with, respectively, three-sixths and four-sixths terpenes, phenolics, leaf toughness, and available foliar of the genera exhibiting significant relationships in the protein. Moreover, it is clear that different genera are predicted direction (Fig. 3, see Appendix D). The two relying on different defense strategies, as each of the six genera that invested in terpenes, Protium and Oxandra, genera had a distinct defense investment pattern (see exhibited very different patterns of terpene investment in Appendix C). For example, only two genera, Oxandra their white-sand and clay specialists (see Appendix D). and Protium, contained measurable terpenes identified Oxandra xylopiodies, the clay specialist, had significantly by GCMS (Appendix C). Similarly, only two genera, higher sesquiterpene and total terpene concentrations Pachira and Parkia had white-sand specialists with than O. euneura, the white-sand specialist (P , 0.05, obviously tougher leaves than clay specialists. The pattern of high phenolic investment and low available Tukey tests; see Appendix D). In contrast, Protium foliar protein in white-sand specialists was more white-sand specialists had higher monoterpene and total consistent across the six genera, but still there were terpene concentrations than did Protium clay specialists exceptions (Oxandra and Protium for phenolics, Mabea (P , 0.05, Tukey tests; see Appendix D). Both Oxandra for available protein; Fig. 3). and Protium white-sand species had significantly higher Whereas different genera invest in different defensive concentrations of diterpenes and other resins than did strategies, we found no consistent relationship between their respective clay specialists (see Appendix D). any particular defensive traits that would suggest either There was no overall effect of origin on leaf toughness a negative trade-off or a synergistic relationship between (see Appendix D). In contrast, white-sand species had defense types (Fig. 3). lower available protein in their leaves than clay special- Amount of defense investment.—We found that five- ists (significant effect of origin, F1, 292 ¼ 393.5, P , sixths of the genera have a higher defense index (DI) in 0.0001; see Appendix D). S156 PAUL V. A. FINE ET AL. Ecology Special Issue

FIG. 1. The effect of origin and habitat in the reciprocal-transplant experiment for (a) the effect of herbivore protection on leaf growth (cm2/d), (b) the effect of herbivore protection on height growth (cm/d), (c) total terpenes and resins (mL terpenes/mg of dry leaf material), (d) total phenolics (g phenolics/g dry leaf material), (e) phenolic : protein ratio (phenolics divided by available protein, a unitless ratio), (f) leaf toughness (grams of mass to punch a 3-mm rod through a leaf; 1.0 g ¼ 1.38 kPa), and (g) available protein (g soluble protein/g dry leaf material). Histograms show means 6 SE. Different letters above bars indicate signiﬁcant differences among the different groups (Tukey tests).

Defensive traits and phenotypic plasticity The effect of habitat on leaf toughness was highly There was no significant overall effect of habitat for significant (F1, 388 ¼ 51.6, P , 0.0001; Fig. 1f). Sixteen of terpenes (Fig. 1c). Aside from the outlier behavior by one 17 species measured had greater leaf toughness in white- species, there was no evidence of phenotypic plasticity in sand than clay habitat; three of those were significant (see phenolic investment (Fig. 1d). Swartzia cardiosperma is Appendix C). In contrast, even though nitrogen availabil- the only species of 20 in the experiment that showed a ities differed by more than five times in the two habitats, significant effect of habitat for phenolic : protein ratios there was no significant effect of habitat on available (see Appendix C). protein for either white-sand or clay specialists (Fig. 1g). July 2006 PLANT TRADE-OFFS AND SPECIALIZATION S157

Phylogenetic independence of growth and defensive traits There was evidence for significant phylogenetic dependence for total phenolics (C-stat ¼ 0.34, P , 0.002), terpenes (C-stat ¼ 0.34, P , 0.002), and leaf toughness (C-stat ¼ 0.32, P , 0.012). The defense index (C-stat ¼ 0.23, P , 0.11) and available protein (C-stat ¼ 0.11, P , 0.148) exhibited a trend toward phylogenetic constraint. We found no evidence for phylogenetic constraint in GI (C-stat ¼ 0.07, P , 0.35) and the protection effect index (C-stat ¼ 0.01, P , 0.399), results that in part might reflect an artifact of our design FIG. 2. Mortality results of the 100% defoliation experiment. Bars show average mortality (6SE) for each origin and because our sampling within each genus was limited to habitat combination. Different letters above bars indicate paired white-sand and clay specialists, which maximized significant differences (post hoc tests, studentized t distribution). the variation between closely related species.

DISCUSSION Evaluating the trade-off: growth vs. defense vs. herbivory Habitat differences in herbivore populations The growth index (GI) and the herbivory index (HI) showed a significant positive relationship (all six of the Two separate measures of herbivorous insect com- genera with positive slope, T0.05(1), 6 ¼ 0, P , 0.025; munities found statistically similar diversity and abun- Fig. 4a). There was a significant negative trade-off dance in the two forest types. In addition, a full third of between GI and total DI, with five-sixths of the genera the morphospecies that were collected more than once showing a negative slope (T0.05(1),6 ¼ 1, P , 0.05, occurred in both habitats. These results are likely Wilcoxon paired signed-ranks test; Fig. 4b). Finally, DI explained by the large home range and dispersal showed a significant negative relationship with HI capabilities of many herbivorous insects (Stork 1988), (T0.05(1), 6 ¼ 1, P , 0.05; Fig. 4c). coupled with the fact that most white-sand forest

FIG. 3. The defense index (DI) scores for each genus are plotted, showing the difference between clay (CL) and white-sand (WS) specialists. The three-letter labels of the lines correspond to the genus table below the plot. Black boxes in the table indicate a significantly higher defensive trait for that genus in the white-sand specialist, and shaded boxes indicate a significantly higher defensive trait for the clay specialist (contrary to predictions). The final column shows the DI scores for each genus, with a negative number signifying a score in the direction contrary to predictions. S158 PAUL V. A. FINE ET AL. Ecology Special Issue

insect herbivores indeed range into white-sand forests. Moreover, these patterns are consistent with our herbivory data from the reciprocal-transplant experiment showing that clay specialist seedlings were attacked at similar frequencies whether they were transplanted into clay or white-sand forests (Fig. 1a, b).

Habitat differences in the impact of herbivory We predicted that the impact of herbivory would be greater in a white-sand forest, because it is more difficult for plants to replace the nitrogen lost to herbivores (Coley 1987b, Craine et al. 2003). This prediction was supported by the fact that all plants transplanted into white-sand forest had significantly higher mortality when defoliated than those transplanted into clay forest (Fig. 2). In the defoliation experiment, white-sand specialists suffered a significantly higher mortality rate than did clay specialists (Fig. 2), confirming a key prediction of the growth–defense trade-off that white-sand specialists ought to have more difficulty replacing tissue lost to herbivores (Coley et al. 1985). This differential response to defoliation by species adapted to low-fertility soils vs. species adapted to high-fertility soils was also found in a study in Singapore (Lim and Turner 1996). Thus, when heavily defended white-sand species are defoliated, they lose costly leaves that represent a high percentage of their energy budget. Due to their slow growth rate, they are then unable to compensate, and this in turn increases their mortality rate (Coley et al. 1987b). For this reason the impact of herbivory appears to be substantially greater for plants adapted to low-resource conditions.

Differences in defense investment Type of defense.—Different genera adopted dramatically different defensive strategies. There was a consistent signal of phylogenetic constraint in our analyses of plant defenses, as genus was a significant factor in each defense variable (see Appendices B and D), and tests for phylogenetic independence confirmed this. In terms of terpenes, phenolics, toughness, and low nutrition, there was no consistent ‘‘syndrome’’ of defensive investment in the six genera; instead, each genus allocated to different combinations of these (and presumably other unmeasured) traits. Indeed, there is little theoretical or empirical support for the idea of a FIG. 4. Plots of the six species pairs for (a) growth rate index vs. protection effect index, (b) growth rate index vs. general negative trade-off between types of defensive defense index, and (c) herbivory vs. defense index. The strategies (Koricheva et al. 2004, Agrawal and Fishbein consistency and magnitude of these slopes were used to test 2006). the predictions of the growth–defense trade-off hypotheses. The three-letter labels correspond to the six genera listed in Fig. 3. Amount of defensive investment.—For Protium,we found higher concentrations of terpenes in white-sand habitats in the Iquitos area are only a few square specialists as predicted, but for Oxandra the reverse kilometers. It is important to recognize that our pattern was found (Fig. 3). The terpene profile of herbivore sampling was extremely limited and precludes Oxandra is driven by sesquiterpenes, which could us from drawing definitive conclusions concerning the possibly be serving a function other than defense, or relative abundance of herbivore populations in white- do not function in a dosage-dependent fashion (Ger- sand and clay habitats. Nevertheless, our herbivore shenzon and Croteau 1991, Langenheim 1994). In estimates represent two independent corroborations that contrast to sesquiterpenes, both Protium and Oxandra July 2006 PLANT TRADE-OFFS AND SPECIALIZATION S159 white-sand specialists were found to have higher When the protection effect of each species is plotted diterpenes and other resins compared to clay specialists against the overall growth rate (Fig. 4a), all six genera (see Appendix B). Diterpenes are not volatile and are exhibited a positive relationship. In each genus, herbi- thought to be either toxic (Lerdau and Penuelas 1993) or vores selectively attacked the faster growing species a type of physical defense against herbivores or more than the slower growing species. This is evidence pathogens (Langenheim 1994). that faster growing plants have lower resistance to Total phenolics and phenolic : protein ratios were herbivores, consistent with the predictions of the significantly higher overall for white-sand specialists growth–defense trade-off. Coley (1983, 1987b) found than for clay specialists (see Appendix B). In our study, the same relationship in Panama where the growth rates percentage dry mass of total phenolics ranged 3–37%,a of 40 species of trees were positively correlated with their large range that is certainly an overestimate and rates of herbivory. highlights the difficulty of precise phenolic quantifica- In the graphs of Fig. 4a, the lengths of the lines tion in the laboratory (Appel et al. 2001). Finally, we correspond to the amount of variation in growth rate found significantly less available protein in white-sand and antiherbivore traits within the species (white-sand specialists. This was the most consistent trait, with five- and clay) of each genus. For example, some genera like sixths of the species pairs showing the same pattern (see Parkia are represented by longer lines in the horizontal Appendix D). direction (Fig. 4a), because this genus includes both shade-tolerant species and those that thrive in high-light Defensive traits and phenotypic plasticity conditions. Therefore, the clay specialist in Parkia is a We did not find many cases of phenotypic plasticity in very fast grower relative to the Protium and Swartzia the seedlings’ allocation to chemical defenses. Very few species, all of which are shade-tolerant species and never species had significant increases or decreases in terpenes found in tree-fall gaps (P. V. A. Fine, personal or phenolics due to habitat (Fig. 1c, d). Similarly, observation). Yet the fact that the slopes of the six lines available foliar protein did not change depending on in Fig. 4a are so similar suggests the existence of a where the seedlings were planted (Fig. 1g), even though universal trade-off, even among species with such nutrient levels were significantly different between the disparate growth rates and defensive strategies. two habitats. We conclude that, for the genera in our When the defense index (DI) scores for the six genera study, herbivore resistance due to chemical defenses and are plotted against their growth rate index (GI) (Fig. available protein content is due to genetically based, 4b), we found a significant negative relationship, with fixed traits (but see Boege and Dirzo 2004). Thus, five of the six genera having higher DI scores in the defense differences result from natural selection by slower vs. faster growing species. The slopes in this herbivores and are not just passive responses to differ- graph exhibit much more variation than the growth vs. ences of available nutrients in the soils. herbivory graph (Fig. 4a), likely due to the coarse In contrast to our results with chemical and nutri- method by which we attempted to quantify defense tional defenses, we found a strong overall effect of investment in these species. The one outlier genus, habitat on leaf toughness, which was significant for three Mabea, shows the opposite relationship than the other species (Fig. 1f; see Appendix C). Overall, we found that five genera, with a higher DI score in its faster growing, leaf toughness was significantly higher for white-sand clay specialist. Because the slower growing (white-sand species in only two genera, Parkia (which we were not specialist) Mabea received the least amount of attack able to measure with our penetrometer, but for which from herbivores in the experiment (see Appendix B), it the pattern was obvious) and Pachira. In contrast, two seems likely that it actually is very well defended and we previous studies found that white-sand plants had failed to accurately quantify its defensive investment. significantly tougher leaves than clay plants (Coley One reason for this may be that Mabea is the only genus 1987a, Choong et al. 1992). These studies did not take of the six that produces copious white latex, and we did phylogeny into account, but their results for white-sand not quantify this trait in our comparisons. The and clay species averages were much more divergent herbivory vs. defense graph (Fig. 4c) echoes this point, than ours. One possibility for the discrepancy is that with Mabea the only genus whose DI score does not toughness in these two studies was only measured in the match its herbivory index score. plants’ home habitats. While our results in no way negate the potentially strong selective effect of herbi- Phylogenetic approach to studying vores on sclerophylly, they do suggest that future the growth–defense trade-off comparisons of white-sand and clay species should not Our approach using multiple pairs of congeners from only be controlled for phylogenetic relationships, but ecologically divergent habitats differs from some other also for resource availability. more quantitative approaches that have used data on branch lengths from a phylogenetic tree to test for Evaluating the growth–defense trade-off correlations between particular traits and habitat The evolutionary trade-off between growth and association (Cavender-Bares et al. 2004). In our defense is illustrated by the data graphed in Fig. 4. approach, we ignore branch lengths by design, since S160 PAUL V. A. FINE ET AL. Ecology Special Issue each of our genus pairs includes just one representative phylogenetic constraint in protection effect (i.e., amount from each habitat type. But in terms of comparisons of of herbivory) or growth in the genera. This is almost growth rate, herbivory, and overall defense as it relates certainly due to the fact that the relevant traits that to white-sand and clay specialization, our results confer resistance to herbivores in low-resource habitats indicate that variation in branch lengths among our and faster growth in high-resource habitats are evolu- pairs matters very little: All six pairs exhibit similar tionarily labile and involve quantitative increases and trade-offs (Fig. 4a). Moreover, if this trade-off has a decreases of already-present qualitative traits related to bearing on a plant’s distribution onto white-sand and growth and defense. clay soils, then evidence for it must be present both in the most recently derived specialist pairs as well as in CONCLUSIONS pairs of species that have persisted for millennia in their By manipulating the presence of herbivores, we particular habitats. By contrast, if we were interested in discovered that defense differences interact with edaphic the evolution of particular traits (like phenolics per se), factors to restrict species to their specialized habitats. then inclusion of some estimate of divergence time (and Although the potential for herbivore attack was similar denser sampling within genera) would certainly be in the two habitats, the impact of herbivory on growth warranted. and survivorship was much stronger in white-sand One limitation of the congeneric pair approach is that forest, giving solid evidence of strong selection for one’s sample is limited to genera that include species that effective defense in white-sand forests. Measurements of occur in both of the habitats of interest. It would be defenses confirmed that white-sand specialists have a interesting to compare genera that were restricted to higher overall defense investment, although each genus only white-sand or only clay habitats to see if the expressed a different combination of defensive traits. growth–defense trade-off was evident in comparisons These results confirmed theoretical predictions that with their sister taxa (that were specialists to the species in low resource habitats evolve a higher optimal contrasting habitat). Our way of calculating a defense defense investment. By testing for defense and growth index (DI) works well precisely because defensive traits differences in white-sand and clay specialists within an are phylogenetically conserved between close relatives, explicit phylogenetic framework, our results strengthen allowing for quantitative comparisons of the same the case that the trade-off between growth and defense is qualitative trait. If we used pairs of taxa that were not universal and governs patterns of plant distribution. closely related, it would become much more difficult to This fundamental trade-off, mediated by herbivores, capture the defense allocation of each contrasting represents an important mechanism of plant coexistence species within a DI, although protection effect would that has been largely overlooked in studies of plant still be an appropriate measure for comparison. habitat specialization and niche assembly. Furthermore, Including a phylogenetic context is vital for studies of this interaction between herbivory and resource hetero- the growth–defense trade-off for at least two reasons. geneity should promote divergent selection in plant First, controlling for phylogeny is critical because it growth and defense strategies that increase the potential reduces the noise of interspecific variation that can easily for ecological speciation. obscure the true patterns in the data (Agrawal and Kotanen 2003). For example, there is substantial ACKNOWLEDGMENTS variation in both growth and herbivory rates among We thank the Peruvian Ministry of Natural Resources these six species pairs (Fig. 4a). Indeed, if phylogenetic (INRENA) for permission to conduct this study; D. Del Castillo, L. Campos, E. Rengifo, and S. Tello of the Instituto de relationships are ignored and one plots all 12 species Investigaciones de la Amazonı´ a Peruana (IIAP) for logistical averages for growth and herbivory together, the support and permission to work in and around the Estacio´ n correlation between growth and herbivory disappears. Allpahuayo; E. Aquituari, M. Ahuite, J. Guevara, M. Jackson, Such an analysis treats each species’ average for growth M. Olo´ rtegui, J. Reed, and F. Vacalla for field assistance; P. rate and defense as an independent data point, an Evans, J. Becerra, and M. Lott, for advice on terpene analyses; J. Heath, K. Pickering, and C. Cohen for assistance in the assumption that is clearly not valid (Harvey and Pagel Appel/Schultz lab; J. A´ lvarez, L. Bohs, D. Dearing, D. Feener, 1991). R. Foster, T. Kursar, and S. Schnitzer, for advice during the Second, it allows one to make direct inferences about entire project; and A. Agrawal, M. Ayres, S. DeWalt, G. Paoli, the phylogenetic patterns of plant defensive traits and and an anonymous reviewer for improving the manuscript. how they relate to habitat specialization. For example, Support was provided by an NSF Predoctoral Fellowship to P. V. A. Fine, an NSF Doctoral Dissertation Improvement Grant terpenes, phenolics, and leaf toughness in our genera to P. V. A. Fine and P. D. Coley, the Michigan Society of exhibit strong signals of phylogenetic constraint. But, Fellows to P. V. A. Fine, an NSF Long-term Research in since species within those genera have a diverse group of Environmental Biology Grant to J. C. Schultz, and NSF grant defensive options, this apparent lack of evolutionary DEB 0234936 to P. D. Coley. lability to completely turn on or off investment into a LITERATURE CITED particular class of defense does not result in lineages Abouheif, E. 1999. 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APPENDIX A Detailed methods for the chemical analysis of terpenes, phenolics, and soluble protein (Ecological Archives E087-117-A1).

APPENDIX B A table presenting all ﬁxed-factor ANOVAs conducted on the growth and defense variables (Ecological Archives E087-117-A2).

APPENDIX C A table presenting growth, herbivory, and defensive traits measured in the experiment for each species in the two soil types (Ecological Archives E087-117-A3).

APPENDIX D Figures showing the effect of origin (white-sand vs. clay specialists) and the genus 3 origin interaction for (a) leaf growth, (b) height growth, (c) the effect of herbivory on leaf growth (protection effect), (d) protection effect on height growth, (e–j) chemical defenses, (k) leaf toughness, and (l) available foliar protein (Ecological Archives E087-117-A4). ENDPLATES. (Top) A British mesotrophic grassland (meadow) plant community. Plant diversity can be divided into a hierarchy of special components, which correspond to a hierarchical set of niches. For an exploration of the correspondence between ecological and evolutionary hierarchies, see the article by Silvertown et al. (pp. S39–S49). (Bottom) Larval monarch butterﬂy (Danaus plexippus)onAsclepias californica. This plant species has high levels of latex and trichomes, which reduce feeding by monarchs and form the basis for one of the milkweed defense syndromes (see the article by Agrawal and Fishbein, pp. S132–S149).