Convergence and Correlations Among Leaf Size and Function in Seed Plants: a Comparative Test Using Independent Contrasts1

American Journal of Botany 86(9): 1272±1281. 1999.

CONVERGENCE AND CORRELATIONS AMONG LEAF SIZE AND FUNCTION IN SEED PLANTS: A COMPARATIVE TEST USING INDEPENDENT CONTRASTS1

DAVID D. ACKERLY2,4 AND PETER B. REICH3

2Department of Biological Sciences, Stanford University, Stanford, California 94305; and 3Department of Forest Resources, University of Minnesota, St. Paul, Minnesota 55108

Prior studies of a broad array of seed plants have reported strong correlations among leaf life span, speci®c leaf area, nitrogen concentration, and carbon assimilation rates, which have been interpreted as evidence of coordinated leaf physiological strategies. However, it is not known whether these relationships re¯ect patterns of evolutionary convergence, or whether they are due to contrasting characteristics of major seed plant lineages. We reevaluated a published data set for these seven traits measured in over 100 species, using phylogenetic independent contrasts calculated over a range of alternative seed plant phylogenies derived from recent molecular systematic analyses. In general, pairwise correlations among these seven traits were similar with and without consideration of phylogeny, and results were robust over a range of alternative phylogenies. We also evaluated relationships between these seven traits and lamina area, another important aspect of leaf function, and found moderate correlations with speci®c leaf area (0.64), mass-based photosynthesis (0.54), area-based nitrogen (Ϫ0.56), and leaf life span (Ϫ0.42). However, several of these correlations were markedly reduced using independent contrasts; for example, the correlation between leaf life span and lamina area was reduced to close to zero. This change re¯ects the large differences in both these traits between conifers and angiosperms and the absence of a relationship between the traits within these groups. This analysis illustrates that most interspeci®c relationships among leaf functional traits, considered across a broad range of seed plant taxa, re¯ect signi®cant patterns of correlated evolutionary change, lending further support to the adaptive interpretation of these relationships.

Key words: comparative method; correlated evolution; independent contrasts; leaf life span; leaf size; photosynthesis; seed plants; speci®c leaf area.

Research on a broad array of terrestrial seed plants has not generally observed: high assimilation rates in long- revealed strong relationships among a suite of leaf func- lived leaves and low rates in short-lived leaves. The for- tional traits, in particular leaf life span, speci®c leaf area, mer combination would appear to be highly adaptive leaf nitrogen concentrations, carbon assimilation rates, (high carbon gain sustained over long periods of time), and leaf conductance (Reich, Walters, and Ellsworth, but may be impossible as leaf longevity may require both 1992, 1997; Reich et al., 1999). In general, species with thickness to resist damage, creating internal self-shading, short leaf life span exhibit high speci®c leaf area, mass- which reduces assimilation, as well as investment in pro- based nitrogen concentration, and carbon assimilation tective physical or chemical traits, which reduces resourc- rates; in log-log analyses, these relationships are linear es invested in the photosynthetic apparatus. In contrast, with r2 Ͼ 0.5 over a broad range of values. This suite of a species with short-lived leaves and low assimilation attributes is also broadly associated with high allocation rates would presumably be eliminated by selection due to leaves, rapid growth rates, rapid attainment of repro- to low growth and fecundity. Optimality models, based ductive age, and regeneration in high resource or dis- on maximizing carbon gain and plant growth, also predict turbed environments (Bazzaz, 1979; Reich, Walters and an inverse relationship between assimilation and leaf life Ellsworth, 1992; Cornellison, Castro-Diez, and Carnelli, span (Kikuzawa, 1991; Ackerly, 1999b). It is important 1998; van der Werf et al., 1998). Recently, it has been to note, however, that up to threefold variation in phys- found that the scaling functions among leaf traits (slopes iological traits may be observed at a particular level of of log-log relationships) are similar across a range of con- leaf life span, so correlations among these traits may be trasting biomes representing a diverse array of conifer weak or nonapparent in samples that do not span a great and angiosperm taxa (Reich, Walters, and Ellsworth, range of values (cf. Diemer, KoÈrner, and Prock, 1992; 1997). These relationships have been interpreted as evi- Reich, 1993). dence of coordinated leaf physiological strategies, re¯ect- In addition to these patterns in leaf physiological traits, ing adaptive and/or biomechanical constraints. For ex- considerable attention has been focused on the ecological ample, the inverse relationship between leaf life span and signi®cance of leaf size variation (Parkhurst and Loucks, assimilation rate indicates that two trait combinations are 1972; Givnish and Vermeij, 1976; Chiariello, 1984). Leaf size directly affects light interception, light penetration 1 Manuscript received 30 October 1998; revision accepted 18 Feb- through the canopy, and leaf energy balance, and leaves ruary 1999. are often smaller in species occupying habitats with high The authors thank L. Onaga and B. Blackman for assistance research- light, low nutrients, or low moisture availability (e.g., Ha- ing phylogenetic resolutions of taxa in this study, and B. Bennett for assistance with programming. Carl Schlichting provided constructive mann, 1979; KoÈrner et al., 1989; Niinemets and Kalevi, comments which improved the manuscript. 1994; see references in Givnish, 1987), conditions that 4 Author for correspondence (e-mail: [email protected]). are also associated with thick leaves, low nitrogen con- 1272 September 1999] ACKERLY AND REICHÐPHYLOGENY AND LEAF FUNCTION IN SEED PLANTS 1273 centrations, and low photosynthetic rates (Reich, Walters, is particularly compounded in comparisons between a and Ellsworth, 1992). In cool-temperate forests, it has continuous and a categorical variable (e.g., seed size vs. also been suggested that the predominance of conifers is gap/nongap regeneration), as there may be few contrasts due to the association between evergreenness and small, for the categorical trait between related species. Further steeply angled leaves (i.e., conifer needles), which en- loss of power occurs when taxonomic schemes (e.g., hance light penetration and low-temperature canopy pho- Cronquist, 1981) are used to construct a phylogeny, be- tosynthesis (Sprugel, 1989; but see Smith and Brewer, cause of the lack of resolution. 1994). Leaf size also varies considerably among species In contrast to these case studies, meta-analyses of pub- within habitats, partly in association with architectural lished results in both animal and plant ecology suggest traits and reproductive morphology (White, 1983; Midg- that there is often little quantitative difference in the cor- ley and Bond, 1989; Ackerly and Donoghue, 1998). relation coef®cients or regression slopes resulting from However, relationships among leaf size and the physio- cross-species and independent contrast analyses (Ricklefs logical traits listed above have not been systematically and Starck, 1996; Price, 1997; Ackerly, 1999a). These examined. cases primarily involve associations between pairs of Adaptive interpretations of relationships such as these continuous traits (e.g., leaf size and branching density, suggest that the traits have exhibited correlated evolu- body size and home range) combined with the use of tionary changes, as selection combined with biophysical fully resolved phylogenies, both of which maximize the constraints would maintain certain trait combinations as number of available contrasts. Recently Ackerly and outlined above. However, the use of cross-species cor- Donoghue (1998) demonstrated quantitatively that sub- relations in comparative ecology has come under intense stantial discrepancies between cross-species and indepen- scrutiny in recent years, particularly in relation to the dent contrast correlations only arise for traits that are very question of adaptive correlations, due to the potential highly conserved from a phylogenetic perspective, i.e., problem of similarity among related species and the sta- when closely related species are also very similar in the tistical independence of species as data points (Harvey functional and ecological traits of interest. and Pagel, 1991). Though it is not always explicitly stat- The objectives of this study were to reevaluate previ- ed, independence in this context is based on a null model ously published analyses of leaf trait relationships for the of the independence of evolutionary changes (Felsen- functional traits described above, together with previous- stein, 1985). Thus we may pose the question in the fol- ly unpublished data for lamina area, based on the data lowing way: do the trait relationships observed among set for 108 species presented in Reich, Walters, and Ells- contemporary species re¯ect or provide evidence of sta- worth (1997) and Reich et al. (1999). A perusal of the tistically signi®cant patterns of correlated evolutionary data suggests that these relationships hold up across dif- changes leading up to the present day? Alternatively, cor- ferent phylogenetic groups and that most traits exhibit relations among extant species may arise due to a small considerable convergence among the taxa in this study number of initial divergence events, in which biologically (Reich et al., 1999). However, both leaf life span and independent changes in two traits happen to be correlat- lamina area exhibit consistent differences between coni- ed, and these changes are passed on to descendent spe- fers and angiosperms that might result in correlations cies. This will lead to correlations between traits among across species that are not observed in independent con- extant taxa despite the independence of evolutionary trasts. Here, we address the following questions: (1) changes in the past. This question has been addressed in Which of these traits exhibit the highest levels of evo- recent years through the development of a number of lutionary convergence (i.e., similarity among distantly re- statistical techniques that incorporate the phylogenetic related species)? (2) Is there a relationship across species lationships among species in order to estimate the evo- between lamina area and leaf functional traits (i.e., spe- lutionary patterns underlying present-day trait distribu- ci®c leaf area, leaf life span, nitrogen concentration, or tions. The most powerful of these is the method of in- gas exchange rates)? (3) To what extent are trait corre- dependent contrasts, which calculates the standardized lations observed across species similar or different using differences in trait values between sister taxa descended phylogenetically independent contrasts, based on recent from each node of a phylogeny and then evaluates trait molecular phylogenetic analyses of seed plants? (4) What correlations or regressions between these contrasts (Fel- are the consequences of alternative seed plant phyloge- senstein, 1985; Garland, Harvey, and Ives, 1992). This nies, and alternative approaches to resolving uncertainty contrast correlation is quantitatively equivalent to the cor- in these phylogenies, for the conduct of comparative an- relation between the evolutionary changes that have oc- alyses? curred along each branch of the phylogeny (Pagel, 1993). In recent years, a number of proposed trait relation- METHODS ships in plant functional ecology have been reevaluated Leaf traitsÐThe following eight traits were considered in this study: in a phylogenetic context, and in several cases the phy- (1) lamina area (LA), (2) leaf life span (LL), (3) speci®c leaf area logenetically structured correlations have been reported (SLA), (4) mass-based leaf nitrogen concentration (Nmass), (5) area based as considerably weaker and nonsigni®cant (e.g., seed size leaf N (N ), (6) mass-based, light-saturated assimilation rates (A ), in relation to establishment conditions: Kelly and Purvis, area mass (7) area-based assimilation rates (Aarea), and (8) leaf conductance (Gs). 1993; Kelly, 1995; stomatal density in relation to life Methods of data collection for traits 2 through 8 are described in detail form: Kelly and Beerling, 1995). These reexaminations in Reich et al. (1999). Lamina area was measured for the entire leaf for have shown that many data sets may contain only a few simple-leaved species, and for the lea¯et in compound-leaved species. appropriate contrasts from a phylogenetic perspective, Analyses of trait correlations focused on the relationships between leaf greatly reducing the power of the analyses. The problem area and the other seven traits, as these have not been previously re- 1274 AMERICAN JOURNAL OF BOTANY [Vol. 86

TABLE 1. Polytomies arising from pasting the 108 species in this study onto a phylogenetic framework provided by the seed plant consensus phylogenies based on rbcL (Chase et al., 1993; Rice, Donoghue, and Olmstead, 1997) or 18S sequences (Soltis et al., 1997). Polytomies arose either from lack of resolution in the underlying tree or from addition of taxa that were not present in these two analyses; families were assigned to the rbcL tree based on Bremer et al. (1998), and to the smaller 18S tree following Chase et al. (1993), or Bremer et al. (1998) if not present in the Chase analysis. All but four polytomies were resolved using the sources at right. All possible combinations of the remaining four were constructed, resulting in 540 systematically resolved trees for comparative analyses (see text).

N Taxa Source for resolution Families±unresolved in rbcL and 18S 1 Betulaceae, Juglandaceae, Fagaceae Manos and Steele, 1997 2 Clusiaceae, Euphorbiaceae, Salicaceae, Chrysobalanaceae unresolved Families±unresolved in rbcL only 3 Diapensiaceae/Sapotaceae, Ericaceae, Sarraceniaceae Kron, 1996 4 Rhamnaceae, Eleagnaceae, Moraceae/Ulmaceae Swensen, 1996 5 Solanaceae, Oleaceae, Apocynaceae/Rubiaceae Olmstead et al., 1993 6 Celastraceae, Violales, Fabales/Fagales clade unresolved Families±unresolved in 18S only 7 Eleagnaceae, Rhamnaceae, Bombacaceae/Moraceae, Ulmaceae, Fagales Chase et al., 1993; Swensen, 1996 8 Fabaceae/Sapindales/etc., Urticales/Fagales/etc., Zygophyllaceae Chase et al., 1993 Genera 9 Pinaceae: Abies, Larix, Picea, Pinus Tsumura et al., 1995 10 Liliaceae: Erythronium, Trillium, Veratrum Kato et al., 1995 11 Ericaceae: Andromeda, Arctostaphylos, Chamaedaphne, Kalmia, Lyonia, P. Stevens, University of Missouri, Rhododendron, Vaccinium personal communication 12 Asteraceae: Baccharis, Echinacea, Eupatorium, Gutierrezia, Helianthus, Bremer, 1994, 1996; Jansen and Pterocaulon, Silphium Kim, 1996 13 Melastomataceae: Bellucia, Clidemia, Miconia S. Renner, University of Missouri, personal communication 14 Fabaceae: Baptisia, Eperua, Prosopis, Robinia Doyle, 1995; Lavin, 1995 Species 15 Pinus - nine species Schwilk and Ackerly, unpublished data 16 Picea engelmanii, P. glauca, P. mariana Smith and Klein, 1994 17 Vaccinium arboreum, V. corymbosum, V. myrtilus unresolved 18 Protium - three undetermined species unresolved 19 Quercus - six species P. Manos, Duke University, personal communication 20 Populus deltoides, P. fremontii, P. tremuloides Smith and Sytsma, 1990

ported and between leaf life span and the remaining six functional traits pruned to show only the families represented in the ecological data set for comparison. in this study. Families that were present in the ecological data set but not represented in these phylogenetic analyses (four families for rbcL; Phylogenetic treesÐData were collected for 108 species representing 12 families for 18S) were then joined to af®liated taxa based on Chase 78 genera and 45 families of seed plants, distributed over six distinct et al. (1993) for the smaller 18S analysis or, if not present in the Chase biomes. Four species were measured twice independently either within analysis, based on the classi®cation presented by Bremer et al. (1998). or across distinct biomes, resulting in 112 total records (see Reich et The resulting rbcL-based tree had six polytomies for family-level re- al., 1999). lationships, while the 18S-based tree had four family-level polytomies The construction of phylogenetic trees for broad ecological surveys (Table 1). Figure 1 shows the resulting trees for rbcL and 18S, and the such as this poses various dif®culties. First, the species sampled in correspondence between them. Species within each family were then ecological surveys rarely coincide with those used in molecular phy- attached to the tree, with genera within families and species within logenetic analyses, necessitating extensive substitution of taxa based on genera shown as polytomies. The four species with two independent taxonomic af®liation at the genus and family level. Secondly, uncer- measurements were represented as two adjacent branches at the tips of tainty in the phylogeny at various levels (in part due to the substitution the trees. Both trees had six polytomies for generic relationships and process just mentioned) may lead to numerous polytomies, which po- six polytomies at the species level (Table 1). tentially reduce the number of contrasts and the power of statistical At this point, polytomies were resolved in two ways to obtain a range analyses. Finally, the necessity for combining information from various of alternative fully bifurcating trees for comparative analyses. (1) A set sources makes it impossible to obtain consistent branch lengths, in terms of 250 ``randomly resolved trees'' was generated separately for the of the amount of evolutionary time represented along each branch. Rec- rbcL- and 18S-based trees, using the ``randomly resolve current tree'' ognizing these problems, we used the following procedure to generate option in MacClade (Maddison and Maddison, 1992), which resolves a robust set of phylogenetic trees that combine high levels of resolution polytomies while preserving the remainder of the topology. This step with extensive sensitivity testing to examine the consequences of phy- provides a rapid approach to obtaining alternative, fully resolved phylogenetic uncertainty (cf. Donoghue and Ackerly, 1996). This procedure logenies for sensitivity analyses. (2) Alternatively, ``systematically re- is spelled out in detail to provide guidance for future comparative stud- solved trees'' were constructed based on extensive searches of the lit- ies addressing broad samples of seed plants. erature and consultation with experts for various groups to resolve the First, the strict consensus trees from two distinct analyses of seed polytomies as listed in Table 1. Four polytomies remained unresolved plant phylogeny (rbcL: Chase et al., 1993; Rice, Donoghue, and Olm- for the rbcL tree and three of these also for 18S: three species of Vac- stead, 1997; 18S: Soltis et al., 1997) were pruned to show relationships cinium, three species of Protium, four families of the Violales (Chry- among families represented in each analysis. Next, these trees were sobalanaceae, Euphorbiaceae, Salicaceae, and Clusiaceae), and (rbcL September 1999] ACKERLY AND REICHÐPHYLOGENY AND LEAF FUNCTION IN SEED PLANTS 1275

Fig. 1. Family-level relationships for the 45 families present in this analysis, based on consensus trees derived from rbcL (Chase et al., 1993; Rice, Donoghue, and Olmstead, 1997) or 18S (Soltis et al., 1997) sequences. Families connected with a dashed line were not present in the original analyses and were joined to the 18S tree based on the rbcL analysis, and to the rbcL tree following Bremer et al. (1998). Numbers in parentheses after each family indicate the number of species present in the ecological data set. Numbered polytomies correspond to the list in Table 1. only) relationships among Celastraceae, Violales, and a large clade in- on the number of species in each clade such that the total branch lengths cluding Fagaceae and Fabaceae. Polytomies of three and four taxa have from the root to each terminal taxon are equal. three and 15 binary resolutions, respectively, so all possible resolutions Comparative analyses were conducted with ACAP v2 (Ackerly and of these polytomies produced 135 alternative phylogenies for the 18S- Donoghue, 1998), which calculates QVI values and independent con- based tree and 405 alternatives for the rbcL-based tree, resulting in a trasts and their correlations over multiple fully resolved phylogenetic ®nal set of 540 systematically resolved trees for analysis. trees. Independent contrasts represent differences between the trait val- Finally, a set of 250 fully random trees was created using MacClade's ues of two sister taxa. As a result each contrast may be negative or equiprobable trees algorithm, in order to evaluate the potential range of positive depending on the direction chosen to calculate the difference outcomes for the comparative analyses (cf. Maddison and Slatkin, 1991; between taxa, but the same direction must be used for all traits at each Losos and Adler, 1995; Donoghue and Ackerly, 1996). node. For graphical presentation, the contrasts in the trait plotted along the horizontal axis are all made positive by convention, and those for Comparative methodsÐComparisons of relative levels of evolution- the trait on the vertical axis are positive or negative depending on ary convergence were made using the Quantitative Convergence Index whether the changes in the two traits covary positively or negatively, (QVI), introduced by Ackerly and Donoghue (1998) for the analysis of respectively (Garland, Harvey, and Ives, 1992). In addition, due to the continuous characters (convergence is abbreviated ``V'' to avoid con- symmetry arising from the arbitrary direction of contrasts all correlation fusion with the Consistency Index, CI; see Maddison and Maddison, and regression analyses must be centered on the origin. In this paper, 1992). The QVI is based on linear parsimony algorithms for ordered we focus on correlations rather than regressions to avoid a priori as- discrete characters (see Swofford and Maddison, 1987) and is equivalent signment of independent and dependent variables, but it is important to to 1ϪRetention Index (Farris, 1989); values range from 0, for traits in note that these correlations are centered on the origin, not on the means which closely related species are phenotypically most similar, to 1 for of the two variates as is customary. traits in which similar species are distantly related and closely related species are most dissimilar (see Ackerly, 1999a). Trait relationships RESULTS were evaluated based on correlation analyses, using phylogenetically independent contrasts (Felsenstein, 1985) based on the various alter- Convergence IndexÐValues of the Quantitative Con- native phylogenies. In order to evaluate the robustness of the results, vergence Index ranged from 0.450 for leaf size to 0.686 branch lengths were calculated with all lengths assumed to be equal, or for Aarea (Table 2). In contrast, mean QVI values calcu- following Grafen's (1989) method, which assigns branch lengths based lated over 250 fully randomized trees (testing the null 1276 AMERICAN JOURNAL OF BOTANY [Vol. 86

TABLE 2. Summary of convergent evolution measures for the eight characters in this data set. Minimum and maximum values represent the range for all species, in original units. Tree length is the reconstructed total amount of evolutionary change, based on linear parsimony, and QVI is the quantitative convergence index (for all taxa and for angiosperms separately), both based on log-transformed trait values; these were calculated for an arbitrarily selected fully resolved tree based on the rbcL phylogeny. Results for other trees were very similar and are not shown. QVI results calculated over random trees test the null hypothesis that the distribution of values for each trait is independent of phylogenetic relationships (see text).

Species values QVI QVI for random trees (N ϭ 250) Tree length Trait (abbreviation) Units N Minimum Maximum (log units) All Angiosperms Mean Min. Max. Leaf size (LA) cm2 102 0.1 408 34.15 0.450 0.621 0.850 0.742 0.936 Leaf life span (LL) mo 109 1.2 98 22.37 0.493 0.706 0.831 0.708 0.929 Speci®c leaf area (SLA) cm2/g 111 12.1 469 15.15 0.581 0.736 0.866 0.761 0.950

Nmass mg/g 110 6.8 63.6 11.31 0.586 0.619 0.857 0.767 0.938 Ϫ1 Ϫ1 Amass nmol´g ´s 108 11.5 467.9 18.92 0.592 0.708 0.838 0.754 0.928 2 Narea g/m 109 0.71 9.14 10.80 0.667 0.732 0.856 0.760 0.940 Ϫ2 Ϫ1 Aarea ␮mol´m ´s 107 2.92 18.7 11.06 0.686 0.654 0.837 0.745 0.930 Ϫ2 Ϫ1 Leaf conductance (Gs) mmol´m ´s 101 67 2272 15.24 0.637 0.669 0.842 0.746 0.927 hypothesis of no phylogenetic similarity) ranged from convergence is currently being further explored (D. Ack- 0.831 to 0.866, and the lowest observed value across all erly and D. Schwilk, unpublished data). eight characters was 0.708. Therefore, the observed values were signi®cantly lower than null expectation. The low values for leaf size and leaf life span, and to a lesser Leaf size relationshipsÐConifers and angiosperms ex- extent the other traits as well, primarily re¯ect the dif- hibited signi®cant differences for all eight traits (Table ferences between conifer and angiosperm species in this 3). Conifers have smaller leaves with longer life span, study (leaf size mean Ϯ SD ϭ 2.09 Ϯ 2.84 and 90.6 Ϯ lower SLA, Nmass, Amass, Aarea, and Gs and higher Narea. For 85.8 cm2, respectively, Table 3); large differences be- the entire data set leaf size showed signi®cant cross-spe- tween major clades lead to more conserved trait distri- cies correlations with all functional traits except Aarea; the butions, which lower QVI values (see Ackerly, 1999a). strongest relationship was a positive correlation with QVI values were higher within angiosperms considered SLA, indicating that larger leaved species were also thin- separately, reinforcing this conclusion (Table 2; values ner (Table 3, Fig. 2). However, none of these correlations were not calculated for conifers alone because they are were signi®cant among conifer species and only relation- paraphyletic in the rbcL phylogeny). QVI values were ships with SLA and Narea were signi®cant among angio- virtually identical over the 540 alternative phylogenies sperms. This suggests that most of the relationships ob- based on the 18S and rbcL trees (range of Ͻ0.05 between served across all species were due to the overall differ- the lowest and highest values for all eight characters). ences between the two groups. In contrast, correlations Note that for this analysis, we have tested the null hy- between leaf life span and the remaining six functional pothesis that relationships among species are random traits were generally similar in magnitude across all spe- with respect to their functional traits, leading to very high cies and among species of conifers and angiosperms, re- null expectations of convergence levels. In contrast Ack- spectively (Table 3). Interestingly, the rank order of the erly and Donoghue (1998) randomized reconstructed correlation coef®cients with leaf life span was very sim- changes over the branches of the tree, which leads to a ilar in conifers and angiosperms (Spearman Rho ϭ 0.71), null expectation of greater similarity among species, and while there was no correspondence for the leaf size cor- much lower QVI values, as the phylogenetic structure of relations (Rho ϭϪ0.04). Thus, functional correlates with trait evolution is conserved. The difference between these leaf size, though weak in both cases, are distinctive in two approaches to signi®cance testing of evolutionary these two groups.

TABLE 3. Comparison of trait values and cross-species correlations (R) with leaf size and leaf life span, for all species and for all angiosperms and conifers treated separately. Differences between means for conifers vs. angiosperms were signi®cant in all cases (t test, P Ͻ 0.0001, except

for Aarea:P ϭ 0.012). Sample sizes (and corresponding critical values for signi®cance at P ϭ 0.05) were 99-108 for all species correlations (Rcrit ϭ 0.19), ϳ 20 for conifers (Rcrit ϭ 0.44) and ϳ 85 for angiosperms (Rcrit ϭ 0.21), depending on missing values (see data table in Reich et al., 1999).

Trait means Ϯ 1 SD (log10-transformed) Correlations with leaf size Correlations with leaf life span Trait (abbreviation) Conifers Angiosperms All Conifers Angiosperms All Conifers Angiosperms Leaf size (LA) 0.019 Ϯ 0.533 1.681 Ϯ 0.624 Ð Ð Ð Ð Ð Ð Leaf life span (LL) 1.529 Ϯ 0.373 0.885 Ϯ 0.415 Ϫ0.424 Ϫ0.145 0.042 Ð Ð Ð Speci®c leaf area (SLA) 1.616 Ϯ 0.259 2.063 Ϯ 0.243 0.644 0.080 0.412 Ϫ0.754 Ϫ0.736 Ϫ0.620

Nmass 1.074 Ϯ 0.106 1.284 Ϯ 0.218 0.293 Ϫ0.030 Ϫ0.008 Ϫ0.770 Ϫ0.548 Ϫ0.748 Amass 1.466 Ϯ 0.271 2.009 Ϯ 0.286 0.538 Ϫ0.147 0.183 Ϫ0.885 Ϫ0.761 Ϫ0.852 Narea 0.433 Ϯ 0.218 0.227 Ϯ 0.165 Ϫ0.564 0.002 Ϫ0.536 0.230 0.533 Ϫ0.095 Aarea 0.805 Ϯ 0.161 0.941 Ϯ 0.177 0.113 Ϫ0.225 Ϫ0.203 Ϫ0.600 Ϫ0.392 Ϫ0.574 Leaf conductance (Gs) 2.216 Ϯ 0.230 2.540 Ϯ 0.272 0.430 Ϫ0.261 0.193 Ϫ0.551 Ϫ0.683 Ϫ0.372 September 1999] ACKERLY AND REICHÐPHYLOGENY AND LEAF FUNCTION IN SEED PLANTS 1277

of Ϫ0.424 across species disappeared completely using independent contrasts (see Fig. 2). This occurred because the interspeci®c correlation re¯ects the differences in both traits between angiosperms and conifers (Fig. 3B), as re¯ected in their low QVI values. In the analysis of independent contrasts, these differences are revealed in the large contrast between the Pinaceae branch and the angiosperms, while there is no signi®cant correlation between these traits within each of these groups (Fig. 3E). In contrast to SLA, leaf size was similar for angiosperms across the entire range of leaf life span, and leaves were larger than comparable conifers.

Sensitivity to the phylogenyÐAll correlations of independent contrasts were extremely similar based on equal branch lengths or branch lengths adjusted following Grafen (1989). The results were also very robust over the alternative phylogenies constructed for this analysis. For example, for leaf life span vs. speci®c leaf area, the mean correlations over the systematically resolved trees based on rbcL (N ϭ 435) and 18S (N ϭ 105) were Ϫ0.64 and Ϫ0.62, respectively, with a range of Ϯ0.005 in each case (Table 4). Results were only slightly more variable in the two sets of 250 randomly resolved trees, with slightly Fig. 2. Scatterplot of interspeci®c correlations vs. independent con- lower means and a range of Ϯ0.05. Similar patterns were trast correlations, based on a fully resolved rbcL-based phlogeny for the species in this analysis. Closed circles: correlations between leaf observed for leaf life span vs. leaf size. These analyses size and the seven other traits (abbreviations follow Table 2); open were also repeated on an angiosperm phylogeny based circles: all other pairwise correlations among the seven functional traits. on a combined analysis of rbcL sequences and morphol- Critical values for signi®cance for both correlations are approximately ogy (Nandi, Chase, and Endress, 1998), which appeared R Ͼ 0.19 and R ϽϪ0.19, based on N ഠ 100. after this study had been completed, and the results were very similar to the rbcL-based analyses reported here (results not shown). In contrast, the average evolutionary Phylogenetic correlationsÐResults of evolutionary correlations over random trees were virtually identical to correlations using independent contrasts are presented for the cross-species correlation (see Abouheif, 1998). For one systematically resolved rbcL-based tree; results over leaf life span vs. SLA, the results over random trees bare- alternative trees were generally similar (see below). ly overlapped the rbcL and 18S results, while for life Overall, evolutionary correlations were similar in mag- span vs. leaf size the results were completely divergent, nitude to the ahistorical, interspeci®c correlations (Fig. as expected due to the large differences between the 2). This was especially true for all of the pairwise cor- cross-species and independent-contrast results in this relations involving the seven functional traits, excluding case. leaf size. Figure 3 illustrates three of the pairwise correlations, with the scatterplot of species values on the left, DISCUSSION and of independent contrasts on the right, plotted following the conventions described in the Methods. The cor- The objective of this paper was to reexamine interspe- relation between leaf life span and speci®c leaf area was ci®c correlations among leaf physiological traits in the Ϫ0.754 between species and Ϫ0.642 for independent context of phylogenetic relationships among species. The contrasts (N ϭ 108, both P Ͻ 0.001; Fig. 3A, D). As primary result was to demonstrate that these relationships noted above, the negative relationship between these are generally quite robust and that previous analyses and traits was observed both within and between the angio- interpretations based on cross-species patterns (e.g., sperms and conifers, and angiosperms with long leaf life Reich, Walters, and Ellsworth, 1997; Reich et al., 1999) span also had low SLA values, similar to the values for are supported from a phylogenetic perspective. In addi- comparable conifers (Fig. 3A). The relationship between tion, there were weak associations among leaf size and leaf size and SLA was also signi®cant based on indepen- function, but several of these were primarily due to dif- dent contrasts (N ϭ 102, R ϭ 0.323, P Ͻ 0.001; Fig. 3C, ferences between conifers and angiosperms, not to rela- F). The most signi®cant discrepancies in interspeci®c vs. tionships observed within each of these groups. The anal- independent contrast analyses involved relationships ysis of quantitative convergence levels also demonstrates among leaf size and several other ecophysiological traits. signi®cant phylogenetic structuring of these physiological Four of these relationships (with LL, Nmass, Amass and Gs), traits, especially leaf size and leaf life span (Table 2). which would be considered signi®cant based on cross- This illustrates that even in this phylogenetically diverse species analyses were not signi®cant based on indepen- set of species there is a pattern of functional similarity dent contrasts (Fig. 2, critical value for signi®cance is among related species. This pattern is primarily driven ϳ0.2 in all cases). In particular, in the association be- by the overall similarities among conifers and angio- tween leaf size and leaf life span, a negative correlation sperms, relative to the divergence between these two 1278 AMERICAN JOURNAL OF BOTANY [Vol. 86

Fig. 3. Scatterplots of species trait values and independent contrasts of leaf life span vs. speci®c leaf area (A, D), leaf life span vs. leaf size (B, E), and leaf life span vs. speci®c leaf area (C, F). Independent contrasts were calculated based on an arbitrarily selected rbcL-based phlogeny. Each contrast represents the difference in the corresponding trait values between two sister taxa. Correlation analyses using independent contrasts are calculated through the origin, because the sign of each pair is arbitrary (see text for details). Open symbols: species values or contrasts for angiosperms; closed symbols: values for conifers; cross: contrast between the Pinaceae branch and angiosperms (see Fig. 1). groups; QVI values within angiosperms (Table 2) indicate temperature (Givnish, 1987), and it was suggested that considerable convergence in this subset of ¯owering parallel variation in leaf ecophysiology would lead to as- plant species. sociations among these traits. This hypothesis is partially Various studies have documented interspeci®c leaf size supported by these data, in particular by the positive cor- variation both within and across habitats in association relation among angiosperm species and among indepen- with light levels, nutrient and moisture availability, and dent contrasts for the entire data set between leaf size and

TABLE 4. Sensitivity of trait correlations analyzed over alternative phylogenies (cf. Fig. 3). Phylogenetic relationships among families were based on rbcL or 18S analyses; polytomies in family, generic, and speci®c relationships were resolved systematically, following Table 1, or at random in MacClade. Correlations of independent contrasts were calculated using equal branch lengths.

Leaf life span vs. SLA Leaf life span vs. leaf size (N ϭ 108) (N ϭ 102) Phylogenetic trees used for correlation Mean Minimum Maximum Mean Minimum Maximum Interspeci®c correlation Ϫ0.754 Ϫ0.424 Independent contrasts rbcL Systematic (435) Ϫ0.640 Ϫ0.645 Ϫ0.634 Ϫ0.005 Ϫ0.018 0.006 Random (250) Ϫ0.620 Ϫ0.684 Ϫ0.563 Ϫ0.002 Ϫ0.058 0.047 18S Systematic (105) Ϫ0.620 Ϫ0.625 Ϫ0.614 0.002 Ϫ0.014 0.011 Random (250) Ϫ0.592 Ϫ0.650 Ϫ0.529 0.005 Ϫ0.077 0.048 Random trees (250) Ϫ0.752 Ϫ0.811 Ϫ0.648 Ϫ0.417 Ϫ0.562 Ϫ0.272 September 1999] ACKERLY AND REICHÐPHYLOGENY AND LEAF FUNCTION IN SEED PLANTS 1279 speci®c leaf area, i.e., smaller leaves tended to be thicker. resolutions of uncertain portions of the phylogeny (cf. Of all the traits, leaf size shows the greatest variation Losos and Adler, 1995) are superior to the treatment of among communities in this study, with the smallest mean each polytomy as a single contrast (Pagel, 1992), which leaf size in the dry New Mexico site, and the largest results in a rapid loss of degrees of freedom and power. mean size in Venezuelan rain forest (cf. Reich et al., To facilitate the construction of phylogenies for compar- 1999). However, the species in this study were not sam- ative analyses, a simple utility has been written that pled to compare mean trait differences between com- prunes and pastes branches in order to partially automate munities, and it is possible that random sampling of spe- this process (D. Ackerly and B. Bennett, unpublished cies from contrasting habitats would show stronger as- data). Although much remains to be learned about seed sociations among leaf size and ecophysiological traits plant phylogeny, the current state of knowledge coupled than those observed here. On the other hand, it is under- with these approaches to phylogenetic uncertainty pro- standable that leaf size and function relationships, which vide ample basis to conduct phylogenetically structured re¯ect parallel responses to abiotic conditions, will be analyses of ecological data. The rationalization that phy- weaker than the functional relationships among speci®c logenies are still too crude for these analyses is not jus- leaf area, assimilation, nitrogen, and leaf life span, which ti®ed. re¯ect adaptive and/or physiological constraints on co- The results of this study also illustrate that in many ordinated leaf function. Leaf size exhibits considerable cases the results of phylogenetically structured correla- variation among ecologically similar species and may be tion analyses are quite similar to traditional cross-species associated with other aspects of plant function. For ex- correlations (Fig. 2; cf. Ackerly, 1999a). However, it is ample, in tropical rain forests variation in leaf size among very important to pay attention to those cases in which pioneer and late-successional species is much greater than results of the two approaches are quantitatively different average differences between these groups (Ackerly, from each other, especially when the difference is not due 1996). Leaf size is strongly associated with canopy ar- to a loss of power as discussed in the Introduction. In chitecture and branching morphology, and to a lesser ex- this study we found an example of such divergence in tent with reproductive morphology (White, 1983; Midg- the relationship between leaf life span and leaf size (Fig. ley and Bond, 1989; Ackerly and Donoghue, 1998), and 3B, E). The negative relationship between these two traits these traits may in¯uence leaf size evolution indepen- results entirely from the divergence between the angio- dently of ecophysiological function. Despite the long- sperms and the conifers, and there is no relationship ob- standing interest in leaf size, variation among species served within either group nor in the analysis of inde- within habitats is still poorly explained (cf. Givnish, pendent contrasts for the entire set of species. How 1987). should this difference in the results be interpreted? One The results of the comparative analysis of leaf func- view is that evolutionary changes in trait values along tional traits do provide strong additional support for particular phylogenetic lineages may occur for many rea- broad patterns of convergence in leaf trait correlations. sons or even at random, and the fact that both leaf size In addition, this study highlights several methodological and leaf life span exhibit a shift along the branch leading points regarding comparative analyses of functional and to the angiosperms does not provide statistical evidence ecological traits. As in many comparative ecological of a biological association between the two traits. It rep- studies, the species in this data set are drawn from a resents a single event, so there is no support for a re- broad phylogenetic spectrum. In this analysis, we con- peated pattern of correlated evolutionary change. From structed highly resolved ``supertrees'' for these species this perspective, the correlation observed at the species by substituting the species of interest onto appropriate level might be considered spurious and biologically locations of seed plant phylogenies based on rbcL and meaningless, and the results of cross-species analyses 18S sequences, pruning extraneous portions of the trees, positively misleading. From another view, the changes in and then resolving the numerous polytomies based on both of these traits may in fact be related to the shifts in relevant published studies (see Table 1). This relatively physiology and life history accompanying the evolution time-consuming procedure left us with just four unre- of angiosperms (cf. Bond, 1989). Larger laminas and solved polytomies, but even this small number led to over shorter leaf life span of angiosperms may be two func- 500 distinct phylogenies for examination. However, the tional traits associated with increased hydraulic conduc- results of both the convergence and correlation analyses tance, enhanced photosynthetic rates, and rapid growth, were extremely similar over all of these trees, suggesting relative to conifers as a whole. And, as Sprugel (1989) that reasonable results could be accomplished by picking argued, the combination of small leaves and evergreen- any one resolution at random. Donoghue and Ackerly ness in conifers may in fact be related to their success in (1996) also showed that the variations among the nu- cold environments. In this sense, the divergence event merous alternative parsimonious trees of the seed plant between these groups does represent an instance of bio- rbcL phylogeny (Rice, Donoghue, and Olmstead, 1997) logically signi®cant coordinated evolutionary change, have little in¯uence on analyses of trait correlations. which at the very least has in¯uenced the subsequent suc- Of more practical relevance, results of this study were cess of each group. Furthermore, the maintenance of the also fairly similar when the families, genera, and species differences between the groups may re¯ect continued sta- were pasted onto the rbcL and 18S trees and the resulting bilizing selection within each group, rather than ``phy- polytomies were resolved at random in MacClade. This logenetic inertia'' due to lack of genetic variation or di- is a very rapid procedure which provides fully bifurcating rectional selection (Hansen, 1997). 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