Functional Traits and Ecological Strategies of Inga Species and Their Relatives from Two Central American Forests

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Functional Traits and Ecological Strategies of Inga Species and Their Relatives from Two Central American Forests
Palow, Danielle T
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[Gainesville, Fla.]
University of Florida
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Doctorate ( Ph.D.)
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University of Florida
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Committee Chair:
Kitajima, Kaoru
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Judd, Walter S
Mack, Michelle C
Martin, Timothy A
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Subjects / Keywords:
Biology -- Dissertations, Academic -- UF
inga -- ingeae -- legumes -- traits
City of Miami ( local )
Seedlings ( jstor )
Seeds ( jstor )
Nutrients ( jstor )
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Botany thesis, Ph.D.


My dissertation addressed how functional traits of coexisting plant species vary in relation to habitat preference and seedling regeneration strategies within the species-rich genus Inga, and other species of tropical trees within the tribe Ingeae (Fabaceae), in a wet forest in Costa Rica and a moist forest in Panama. In the first project, I compared functional traits of nine Inga species and their soil type preference in the wet forest, in which a transect-based survey demonstrated their distributional bias to either fertile alluvial soil or less fertile residual soil of volcanic origin. Compared to alluvial soil specialists, sapling and adult leaves of residual soil specialists had higher values of leaf mass per area and lamina density but lower concentrations of nitrogen (N) and phosphorus (P). Despite lower leaf N concentration, residual-soil specialists had higher values of seed mass and seed N concentration, suggesting the potential importance of maternal nutritional provisioning for successful seedling establishment on less fertile soil. In the second project, I examined the relationships among seed and seedling traits among 23 species in Ingeae from the two forests. Large-seeded species transferred a lower proportion of seed mass to the seedling during the seed-reserve dependent stage. Seed mass, however, was uncorrelated with other seed and seedling traits examined. Seed N and P concentrations were correlated with N and P in seedling stems but not in leaves. Thus, certain trait correlations observed across a broad range of phylogeny, such as a negative correlation between seed N concentration and seed mass, may be obscure when closely related species are compared. In the last project, I examined how seedlings of an emergent tree, Balizia elegans, adjust their growth, nutrient and biomass allocation when soil N availability is limited. Lack of soil N resulted in higher biomass allocation to roots relative to leaves. Yet, across all treatments, leaves had the highest pool of N. These results show that functional traits of adult and juvenile leaves, seeds, and seedlings exhibit substantial variation and trait-trait associations among closely related species, at least partially in relation to soil type preference in Neotropical forests. ( en )
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Thesis (Ph.D.)--University of Florida, 2012.
Adviser: Kitajima, Kaoru.
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by Danielle T Palow.

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2 2012 Danielle Therese Palow


3 To all the baby trees that gave their liv es in the pursuit of science; my mom, Diana Travieso Palow, who supported me through all the trials of this degree ; and m y grandmother, Ligia Travieso, whose love of plants I carry on


4 ACKNOWLEDGMENTS I thank my graduate committee, Kaoru Kitajima, Tim Martin, Michelle Mack and Walter Judd, for their support and guidance I also thank the staff of La Selva Biological sta tion, particularly Deedra McClearn and Orlando Vargas, for thei r assistance with logistics and advice while in the field. As well, I thank Nelson Zamora for assistance with species identifications, Deborah Clark, David Clark and Steven Oberbauer for guidan ce with experimental design and general advice and Robin Chazdon for loaning me equipment I thank David Janos for introducing me to Costa Rica and the world of the tropical biolog y I could not have finished my lab work without the assistance of Julia Re iskind and Grace Cummer. I am grateful for my friends, particularly Nalo McGibbon, Melissa Webb, Kara Cohen Host, Amanda Wendt, Erin Kuprewicz, Arietta Fleming Davies, Camila Pizano and Martijn Slot for their help and continu ed support during this process As well, Sara Pinzon assisted me with seed collection in Panama. And lastly I thank Kristen Nolting her assistance with Chapter 2. This research was funded by the South East Alliance for Graduate Education and the Professoriate, the Organization of Tropi cal Studies, and the National Science


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 FUNCTIONAL TRAITS AND SOIL FERTILITY PREFERENCE OF INGA SPECIES IN A WET TROPICAL FOREST ................................ ............................. 18 Background ................................ ................................ ................................ ............. 18 Methods ................................ ................................ ................................ .................. 21 Site, Species and Spatial Distribution ................................ ............................... 21 Functional Trait Measurements ................................ ................................ ........ 22 Statistical Analysis ................................ ................................ ............................ 24 Results ................................ ................................ ................................ .................... 24 Species Distribution ................................ ................................ .......................... 24 Functional Traits ................................ ................................ ............................... 25 Discussion ................................ ................................ ................................ .............. 26 Habitat Association ................................ ................................ ........................... 26 So il Type Preference and Functional Traits ................................ ...................... 27 Ontogenetic Changes in Leaf Traits ................................ ................................ 29 Seed Traits ................................ ................................ ................................ ....... 30 Summary ................................ ................................ ................................ .......... 31 3 SEED AND SEEDLING TRAIT COVARIATIONS AMONG SPECIES IN INGEAE (FABACEAE) FROM TWO NEOTROPICAL FORESTS ........................... 36 Background ................................ ................................ ................................ ............. 36 Methods ................................ ................................ ................................ .................. 39 Study Sites ................................ ................................ ................................ 39 Study Genera ................................ ................................ ............................. 40 BCNM Study ................................ ................................ .............................. 41 LS Study ................................ ................................ ................................ .... 41 Statistical Analysis ................................ ................................ ..................... 43 Results ................................ ................................ ................................ .................... 43 Discussion ................................ ................................ ................................ .............. 45 Summary ................................ ................................ ................................ ................ 48


6 4 EFFECTS ON SEED RESERVE AND SOIL NITROGEN ON EARLY SEEDLING GROWTH OF BALIZIA ELEGANS ................................ ...................... 57 Background ................................ ................................ ................................ ............. 57 Methods ................................ ................................ ................................ .................. 59 Results ................................ ................................ ................................ .................... 61 Discussion ................................ ................................ ................................ .............. 61 5 CONCLUSIONS ................................ ................................ ................................ ..... 68 APPENDIX A ADDITIONAL TABLES AND FIGURE FROM CHAPTER 2 ................................ .... 71 B ADDITIONAL TABLE AND FIGURES FROM CHAPTER 3 ................................ .... 74 LIST OF REFERENCES ................................ ................................ ............................... 77 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 87


7 LIST OF TABLES Table page 2 1 Distribution of Inga species along 104 5 0m long trail segments at La Selva Biological Station for which at least a total of 10 individuals taller than 50 cm were encountered. ................................ ................................ .............................. 32 2 2 Species means for seed mass, N and P concentrations, and wood specific gravity (WSG) of nine Inga species ................................ ................................ .... 33 2 3 The results of nested 2 way ANOVA testing the effects of soil preference (alluvial or residual), ontogenetic stage (sapling or adult), species (Sp, nested within soil preference type), and their interactions on five leaf traits. .................. 34 3 1 Species mean values of seed mass (g) excluding seed coat, seedling mass (excluding cotyledon) (g) and nitrogen (N) per unit dry mass (mg/g) in seeds and seedling organs.. ................................ ................................ ......................... 50 3 2 Mean phosphorus (P) concentrations (mg/g dry mass) in seeds and seedling organs and relative growth rate (RGR) of seedlings for species sampled at LS. ................................ ................................ ................................ ...................... 51 3 3 Species mean values for cotyledon mass (g), N concentration (mg/g), P concentration (mg/g) and the number of cotyledons attached to the main seedlin g axis at harvest. ................................ ................................ ..................... 52 3 4 correlations between pairs of seed, seedling and adult traits, using the combined data sets fr om both sites. ................................ ................................ ... 53 4 1 solution. ................................ ................................ ................................ .............. 64 4 2 Trait means (standard error) for all treatments and harvests. ............................. 64 4 3 Results of Kruskal Wallis test among nitrogen treatment groups. ...................... 65 A 1 Means of adult t raits by soil type for the three species that were collected on both soil types at La Selva Biological Station. ................................ .................... 71 A 2 Means for leaf and sapling traits of nine Inga species at La Selva Biologica l Station, Costa Rica, including LMA (leaf mass per area), l amina T (lamina thickness), l amina D (lamina dry mass density), h eight (mean adult height), DBH (diameter at breast height), and Narea (leaf N per unit lamina area). ........ 72 B 1 The results from an ANOVA test comparing seed and seedling traits between species and species nested within study site. ................................ .................... 74


8 LIST OF FIGURES Figure page 2 1 Three leaf traits (mean + s.e.) of saplings (left side) and adults (right side) of nine Inga species, listed in the order of strong to lesser bias to residual soil (Tables 1, 2). ................................ ................................ ................................ ...... 35 3 1 Species mean (A) seed N concentration (mg/g) and (B) P concentration (mg/g) plotted against seed mass. ................................ ................................ ...... 54 3 2 Species mean (A) N and (B) P concentration (mg/g dry mass) in seedling organs plotted against N and P concentration in seed excluding seed coat.. ..... 55 3 3 Ratio of (A) the seeding mass to seed mass, and ratio of total seedling nutrient pool size to s eed nutrient pool size of (B) N and (C) P, at the first fully expanded leaf stage for species sampled at LS.. ................................ ........ 56 4 1 Total leaf area (cm 2 ) of B. elegans seedlings (mean se) at different number of days after nutrient solution treatments.. ................................ .......................... 66 4 2 Total seedling biomass (g) of B. elegans seedlings (mean se).. ...................... 66 4 3 Nitrogen c oncentration (%) in organs for seedlings harvested at the beginning (p) and after 44 days under all nutrient (a) and no N (n) treatments.. ................................ ................................ ................................ ......... 67 A 1 Phylogenetic relationships among 19 species in In gae (including nine Inga species in the study plus those in other genera that occur in La Selva). ............ 73 B 1 Mean seed nutrient concentrations (mg/g) plotted against seed mass for each mother tree. ................................ ................................ ............................... 75 B 2 Mean seedling organ N (A) and P (B) concentration (mg/g dry mass) for each mother tree plotted against N concentration in seed excluding seed coat. ......... 76


9 Abstr act of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FUNCTIONAL TRAITS AN D ECOLOGICAL STRATEG IES OF INGA SPECIES AND THEIR RELAT IVES FROM TWO CENTRA L AMERICAN FORESTS By Danielle Therese Palow May 2012 Chair: Kaoru Kitajima Major: Botany My dissertation addressed h ow functional traits of coexisting plant species vary in relation to habitat preference and seedling regeneration st rategies within the species rich genus Inga and other species of tropical trees within the tribe Ingeae (Faba ceae) in a wet forest in Costa Rica and a moist forest in Panama In the first project, I compared functional traits of nine Inga species and thei r soil type preference in the wet forest in which a transect based survey demonstrated their distribution al bias to either fertile alluvial soil or less fertile residual soil of volcanic origin Compared to alluvial soil specialists, sapling and adult lea ves of residual soil specialists had higher values of leaf mass per area and lamina density but lower concentration s of n itrogen (N) and phosphorus (P) Despite lower leaf N concentration, residual soil specialists had higher values of seed m ass and seed N concentration suggesting the potential importance of maternal nutritional provisioning for successful seedling establishment on less fertile soil. In the second project, I examined the relationships among seed and seedling traits among 23 species in Ingeae from the two forests Large seeded species transferred a


10 lower proportion of seed mass to the seedling during the seed reserve dependent stage. Seed mass, however, was uncorrelated with other seed and seedling traits examined. Seed N and P concentrations were correlated with N and P in seedling stems but not in leaves. Thus, certain trait correlations observed across a broad range of phylogeny such as a negative correlation between seed N concentration and seed mass, may be obscure when c losely related species are compared. In the last project, I examine d how seedling s of an emergent tree, Balizia elegans adjust their growth, nutrient and biomass allocation when soil N availability is limited Lack of soil N resulted in h igh er b ioma ss allocation to roots relative to leaves Yet, across all treatments, leaves had the highest pool of N The se results show that functional traits of adul t and juvenile leaves, seeds, and seedlings exhibit substantial variation and trait trait associations among closely related species at least partially in relation to soil type preference in Neotropical forests.


11 CHAPTER 1 INTRODUCT ION Tropical forests are known for their high species richness, and multiple mechanisms have been proposed to explain the maintenance of diversity at local and regional scales (reviewed in Kitajima & Poorter 2008). One such mechanism is ecological niche di fferentiation based on species differences in regeneration niches, susceptibility to natural enemies, seed dispersal limitation, and competitive ability (Burslem, Garwood & Thomas 2001). Chase & Liebold (2003) define a species niche as the environmental co nditions that meet a species minimum requirements such that a separated into the fundamental niche, i.e. the environmental tolerance range in which individuals with in a species can potentially grow and reproduce, and the realized niche, which is narrower due to biotic factors including competition and natural enemies. Hence, ecological niche space is defined in multiple dimensions including environmental factors (e.g ., light, temperature, water, soil nutrients) and biotic interactions (e.g., competition, predation, disease and mutualism). Silvertown (2004) suggest ed niche differentiation along multiple environmental axes plays a more significant role in plant species coexistence than previously thought. In this dissertation I studied niche differentiation of tropical trees in relation to heterogeneity of soil nutrient resources, as well as functional traits of seeds and young seedlings. In an effort to understand the f unctional basis for species differences in ecological niches, plant ecologists have long compared traits among species that differ in their ecological niche specializations, in particular morphological traits that are relatively easy to compare across many species (Shipley 2010). This approach has become more


12 popular in recent years as researchers have been able to compile large databases of plant traits to elucidate general patterns or correlations among traits, for example, the widely cited compilation of leaf traits by Wright et al. (2004) and wood traits by Chave et al. (2009). In the most general sense, a trait is defined as a property of an organism that is measurable at the individual scale without measurements external to the organism, such as enviro nmental data (McGill et al. 2006; Violle et al. 2007). A functional trait is a physio growth, reproduction and survival (Violle et al. organism et al. 2006). Traits that influence resource acquisition for growth (e.g. leaf mass per area; defined as leaf mass/leaf area) or regulation of metabolism (e.g. dark respiration rate) are examples of plant functional traits (Ackerly 2003). Certain leaf traits, such as leaf mass per area, leaf nitrogen concentration and wood density, are considered to be fundamental for plant resource economics, and have been compiled and analyzed at regional and global scales (Wright et al. 2004 Wrig ht et al. 2005; Chave et al. 2009). These traits are relevant not only at the individual scale due to their relationships with growth and/or photosynthesis, but also at the ecosystem scale for primary productivity and nutrient cycling (Shipley et al. 2006) For example, species with high wood density tend to have lower intrinsic growth rates and slower decomposition rates (Chave et al. 2009), whereas species with high leaf nitrogen concentration have higher photosynthetic rates and contribute to rapid nutri ent turnover (Wright et al. 2004; Cornelissen et al. 2004). Certain trait trait correlations have been identified as universal both at local and global scales, for example, the negative


13 correlation between leaf mass per area and leaf nitrogen (N) co ncentra tion (Wright et al. 2004, Wright et al. 2005), and between wood density (dry mass per unit green volume) and leaf size (Wright et al. 2007). Such correlations are believed to exist as a result of trade offs between leaf photosynthetic capacity, constructio n costs, and tissue turn over rates that vary from short lived productive leaves to long lived well defended leaves that slowly pay back their construction cost (Shipley et al. 2006). These trade offs may relate to different life history strategies that ca n lead to coexistence of species (Westoby et al. 2002; Kitajima & Poorter 2008). In conjunction with a general increased interest in plant functional traits, McGill et al. (2006) suggest ed that community ecologists should place high priority on two questio along abiotic environmental gradients and (2) how do biotic interactions determine a speci es to a particular habitat, detectable as the probability of finding a species more often in one habitat over others, can be interpreted as a species realized niche (Weiher et al. 2011). Numerous plant species exhibit habitat association in tropical commun ities (Clark Clark &Read 1998; Baraloto et al. 2007; Queensborough et al. 2007; Russo et al. 2005, 2008). Furthermore, such soil habitat specialization may have promoted speciation, as demonstrated by Fine et al. (2005) for specialization to fertile vs i nfertile soil in the Burseraceae. Furthermore, an analysis of tree species habitat associations in a moist forest in Panama revealed that 51 75% of the species, depending on the statistical test, exhibited habitat associa t i on (Harms et al. 2001). Similarly John et al. (2007) found that 36 51% of tree species in three Neotropical forests showed strong


14 associations to spatial distributions of soil nutrients. While most of these studies did not examine which functional traits may be associated with the observ ed soil habitat specializations, in seasonally flooded and terra firme lowland rain forests of French Guiana, association to soil habitat was found to be linked to differences in leaf traits, such as specific leaf area and photosynthetic capacity (Baralott o et al. 2007). Also recognized is the importance of phylogeny in functional trait variation and ecological niche specialization. Traits may be conserved such that their variation is constrained within phylogenic groups (Ackerly 2003). Wood density of Neot ropical trees, for example, is highly conserved, as intergeneric variation explains 74% of species level variation (Chave et al. 2006). The degree of phylogenetic niche conservatism, however, varies among clades and individual traits (Losos 2008). With adv ances in phylogenetic techniques and analyses during the last 10 years, comparing traits of species in the context of their phylogenetic background or relatedness has become easier (Webb et al. 2002). For example, in community wide comparison across a broa d range of taxa in a wet forest in Amazonian Ecuador, Kraft, Valencia & Ackerly (2008) found that leaf functional traits of species that co occur in a particular topographical habitat were ecologically and phylogenetically more similar than a null model wo uld predict, suggesting phylogenetic constraints in trait habitat associations. In contrast, Cavender Bares, Keen & Miles (2006) showed phylogenetic conservatism of traits in a community level analysis of all tree species, but found trait overdispersion (i .e., a given niche is occupied by less related species than expected by chance) when the analysis was restricted within the genus Quercus which apparently has gone through a rapid adaptive radiation in North Central Florida. Hence, whether traits show ove r dispersion


15 (in which related species are ecologically different) or niche conservatism (in which related species are ecologically similar) may differ depending on the phylogenetic diversity sampled in the study. One advantage of comparing traits of relat ed species is that it allows evolutionary inference about how traits diverged from a common ancestor (Ackerly 2003), making it possible to interpret trait variations as adaptation to contrasting environmental conditions. Conversely, comparing unrelated spe cies makes it difficult to separate the effects of environment and evolutionary history on trait variation. Thus, I chose to compare trait variation of species within a speciose clade of Neotropical trees in the tribe Ingeae in Fabaceae. This is a good mod el system because other ecologists have found that Inga exhibit habitat preference (Harms et al. 2001; Dexter, Pennington & Cunningham 2010 ), Inga species have differences in anti herbivore traits (Kursar et al. 2009) and species of Inga exhibited differen t relative growth rates and leaf traits as juveniles (Palow & Oberbauer 2009). Furthermore, greater u nderstanding of trait variation within Fabaceae (legumes), the third largest plant family and a significant component (>15%) of lowland tropical forest com munities (Burnham & Johnson 2004), will contribute to tropical forest community and ecosystem research. Fabaceae are often considered a unique functional group because of their high nitrogen demand and use, and are important because of the potential they h ave to affect the amount of nitrogen in an ecosystem. The overall objective of my research was to explore patterns of functional traits among related species as a potential mechanism for coexistence in a species rich tropical forest. I conducted three stud ies, two observational and one experimental, to


16 examine various traits and the effects environmental factors and ontogenetic stages have on those traits. I asked three broad questions: (1) How do functional traits differ according to soil type preference? (2) Are there general interspecific correlations among traits of seeds, seedlings, sapling leaves, and adult leaves and wood density? (3) Does soil nitrogen availability affect seedling allocation patterns in predictable ways? In Chapter 2, I examine funct ional traits and soil type preference in the species rich Neotropical genus Inga I quantif y functional traits of both saplings and adults of nine common Inga species. I hypothesized that species exhibiting soil type preference would differ in their functi onal traits according to their soil preference. In other words, I predicted that traits of species that prefer the same soil type would be more similar to each other than to those that prefer another soil type. In addition, I hypothesized that functional t raits would differ with ontogenetic stage within species. To test these hypotheses, I stud y bias in distribution of saplings and adults to two major soil types at La Selva Biological Station (LS), Costa Rica. I compare selected leaf traits, wood density, a nd seed traits that are considered relevant for optimal growth and regeneration strategy in relation to soil fertility. In Chapter 3, I examine seed and seedling traits of several co occurring species in the tribe Ingeae from two Neotropical sites: Barro Colorado Nature Monument, Panama and LS. I use seed mass as a key trait to examine trait variation and compare 23 species. Specifically, the traits I examine are seed nitrogen and phosphorus reserves (concentrations), how seed reserves (biomass, nitrogen a nd phosphorus) are allocated to leaves, stems and roots of developing seedlings, and the proportion of seed reserves transferred from seed to seedling. I hypothesized that resource allocation patterns in


17 seedlings should be correlated with seed size and nu trient concentrations. I test this hypothesis with a shade house study in which I grew seedlings of several species of Ingeae from seed until the first leaf became fully expanded. During this developmental stage, seedling growth and development were expect ed to be largely dependent upon seed reserves, and seedlings can be vulnerable to many hazards (eg. pathogens, trampling, herbivory) as they become established. This i s the first study of how seed and early seedling traits vary as a result of rapid adaptiv e radiation in a species rich legume genus. Finally, in Chapter 4 I examine how nitrogen and biomass are allocated in seedlings of a tropical legume tree, Baliza elegans when they are grow n with and without external nitrogen soil supply. Seedlings were at the transitional stage from seed dependence to complete autotrophy. Even in tropical soils that are not considered to be nitrogen limited, nitrogen may be a limiting resource for seedlings without well developed root systems. I hypothesized that seedlings not receiving nitrogen from the soil would allocate to growth of below ground biomass (roots). To test this hypothesis I grew seedlings in shade houses and gave them nutrient solutions that were either complete or, without nitrate and ammonium. A unique s trength of my research is the focus on functional traits of closely related species at multiple ontogenetic stages. Few studies have explored how traits differ at multiple ontogenetic stages in relation to habitat preference, particularly within a standard ized phylogenetic background. In doing so here I hope to contribute significantly to a growing body of plant functional trait data and improve understanding of their ecological significance.


18 CHAPTER 2 FUNCTIONAL TRAITS AN D SOIL FERTILITY PREFERENCE OF IN GA SPECIES IN A WET TROPICAL FOREST Background Specialization to soil resource niches may be an important mechanism that promotes speciation in tropical forests (Fine et al. 2005), which should go hand in hand with divergence of functional traits that lead to demographic consequences and distributions across habitat heterogeneity (Kraft, Valencia & Ackerly 2008). Such trait based habitat filtering of individuals is indicated in both community wide comparisons (Russo et al. 2005; John et al. 2007) and within family comparisons conducted for Lauraceae, Burseraceae, Malvaceae (Sterculiaceae), Myristicaceae and Fabaceae (Sri Ngernyuang et al. 200 3; Fine et al. 2005; Yamada et al. 2006; Baraloto et al. 2007; Queenborough et al. 2007). In order to link trait diver gence with evolution of habitat preference, the most direct eviden ce could be sought among close relatives that share phylogenetic constraints (e.g., within genus Quercus by Cavend e r Bares Kitajima & Bazzaz 2004). But, functional trait diversification wit hin a genus has rarely been quantified for tropical trees (Queenborough et al. 2007), even though soil habitat specialization is known for species rich genera, such as Inga (Harms et al. 2001; Dexte r, Pennington & Cunningham 2010; Endara & Jaramillo 2011) and Shorea (Russo et al. 2005) that contribute significantly to local tree diversity. Functional traits that affect growth rates and survival vary in relation to specialization to resource niches and ecological strategies of the s pecies (Cornelissen et al. 2003; Cavender Bares et al. 2004; Kitajima & Poorter 2008; Wright et al. 2010). In particular, leaf mass per area (LMA), leaf nitrogen (N) concentration, leaf size, seed mass and wood specific gravity (WSG) have been linked to growth survival trade offs


19 a nd resource niche specialization (Baraloto et al. 2007). Low LMA and high leaf N are typical of fast growing species common in resource rich environments such as treefall gaps and fertile soils (Wright et al. 2004; Poorter & Bongers 2006; Kraft et al. 2008 ; Ordoez et al. 2009). Dry mass density (dry mass per area) of leaves and stems are associated with each other and growth survival trade offs; high values of WSG and lamina density are associated with high survival, slow growth and specialization to shade d or infertil e habitats (Poorter et al. 2008; Russo et al. 2008; Chave et al. 2009; Kitajima & Poorter 2010). The degree of soil habitat specialization increases from juveniles to adults (Russo et al. 2005), but it is not well understood whether environme ntal filters acting upon functional traits operate at either or both the juvenile and adult stages to create the observed patterns of trait environmental associations. Information on how adult and juvenile traits differ in relation to ecological niche spec ialization of species will contribute to mechanistic understanding of the demographic processes that lead to habitat filtering (Lusk & Warton 2007; Poorter 2007 ; Kitajima & Poorter 2010). Seedling establishment represents the earliest ontogenetic stage in which trait based habitat filtering may operate, for which functional traits of seeds are relevant. Larger seeds initially produce larger seedlings that can tolerate various hazards including burial by litter and physical disturbance (Moles et al. 2004), a nd enhance establishment in nutrient poor soils by enabling construction of larger initial root system (Jurado & Westoby1992, Hanley et al. 2007). Furthermore, higher concentration of a mineral element may extend the time during which seed reserves support seedling demands for that nutrient (Hanley & Fenner 1997; Kitajima 2002). However, existing data are equivocal as to the


20 relationship of seed size and mineral nutrient concentration with specialization to fertile vs. infertile soils; tropical species foun d on more infertile soils have smaller seeds with higher N concentrations (Grubb & Coomes 1997), and Proteaceae species from infertile sandstone soils have larger seeds with higher phosphorus (P) concentrations than those from less inf ertile soils (Esler e t al. 1989; Milberg & Lamont 1997; Groom & Lamont 2010). Russo et al. (2007) found no association between seed size and soil habitat specialization for Malaysian tropical tree species, but comparison within a narrow phylogenetic group may be more conclusiv e because phylogenetic constraints are particularly strong for seed size. The objective of this study is to examine how habitat association and ontogeny affect leaf, wood and seed traits of species in the genus Inga in Fabaceae (legumes). Fabaceae are a s pe cies rich family that represent ~16% of the woody species in Neotropical forests (Burnham & Johnson 2004). Inga is a large Neotropical genus consisting of ~300 species that rapidly diversified within the last 2 10 million years (Richardson et al. 2001). Within the wet tropical forest of La Selva Biological Station (LS) in Costa Rica, expert observations suggest that several Inga species appear to grow either on rich alluvial soil or less fertile residual soil that occur in close prox imity (Zamora & Pennin gton 2001; N. Zamora personal communications). We asked the following questions using common Inga species at LS: (1) What is the degree of habitat association of Inga species according to soil type, residual or alluvial, at LS? (2) Do leaf and wood functi onal traits of adults and juveniles differ with the observed soil type preferences? (3) How do juvenile leaf functional traits correlate with adult leaf functional traits? (4) Do residual soil specialists differ in seed mass and


21 nutrient content from alluv ial specialists? We predicted that both adults and juveniles of species associated with less fertile residual soils show trait syndromes associated with slower growth, such as high values of LMA, WSG, and seed size, but low concentrations of N and P compar ed to alluvial soil specialists. Based on other studies that examined juvenile and adult leaf traits (e.g., Poorter 2007; Kitajima & Poorter 2010), we expected that a given leaf trait would exhibit positive correlation between juveniles and adults. Methods Site Species and Spatial Distribution La Selva Biological Station is a tropical wet forest in the central lowlands of Costa ) soils on river terraces, both belong to Oxisols (Kleber et al 2007) with similar total N, pH and water contents, but total nutrient stocks in 0 75 cm depth (kg ha 1 ) are much lower in residual soils than alluvial soils for phosphorus (44% lower) and pot assium (39% lower) (Espeleta & Clark 2007). Distribution of several species of trees and palms at LS exhibit significant associations with either of these soil types (Clark, Palmer & Clark 1999). Zamora & Pennington (2001) classify species within Inga to t hose with no soil type preference (generalists), alluvial soil specialists, residual soil specialists and riparian species, and describe their growth habits as ranging from sub canopy trees to canopy emergents. Twenty species of Inga are found at LS, and m any are considered secondary forest species (Zamora & Pennington 2001). According to the best molecular phylogeny available, there was no obvious phylogenetic bias in soil type preferences among Inga species at La Selva (Fig. A 1; Kyle Dexter unpublished d ata).


22 In order to confirm these expert observations, we surveyed the distribution of all Inga individuals along 52 transects, each 50 m long, per major soil type (residual or alluvial) along existing trails at LS. The trail segments surveyed were randomly chosen from all trail segments within old growth, second growth and selectively logged forest. We recorded all individuals of Inga that were > 50 cm tall and occurred within 2 m of the trail edge, identifying them to species and recording the location of i ndividuals > 2m tall. Voucher specimens of each species were collected and identified by Nelson Zamora at the National Biodiversity Institute of Costa Rica. A selection of these vouchers were left at the National Herbarium of Costa Rica. Functional Trait M easurements For the nine most common species of Inga encountered in our survey, we determined species mean values for leaf traits (LMA, lamina thickness, lamina density, lamina N, lamina P) for adults and juveniles, wood specific gravity (WSG) of adults, s eed biomass, seed N, and seed P. While most individuals sampled occurred within 2 m of an established trail, some individuals were sampled up to 20 m away from an established trail. All were collected from areas where the soil type has been mapped. Leaves and wood were primarily collected from individuals growing on their preferred soil type (as identified by the species distribution survey). We attempted to collect samples from individuals occurring on both their preferred and non preferred soil types to a ssess how trait values may show plastic responses to in situ soil types where individuals were sampled. Most species, however, rarely if ever occurred on both soil types, and we could collect an adequate number of samples only for three species. For the th ree species sampled from the two soil types the traits of individuals did not differ significantly (see Table A 1 ). Leaves from adults (a minimum of five per individual from


23 the preferred soil type) were collected from the canopy using a cross bow to break off small branches from the tree. For saplings (defined as 1 3 m tall), we collected three leaves per individual. Leaves were collected from the highest position of the tree crown, wrapped in wet paper towel along with branches, sealed in plastic bags and kept cool in a refrigerator overnight until they were processed the following day. Light environment of each tree was categorized using a crown illumination index (CII, see Clark & Clark 1992); the amount of the crown exposed to light and the direction of light were estimated. Trait measurements generally followed Cornelissen et al. (2003). Leaf processing included wiping them clean of epiphylls with a paper towel, followed by measurement of leaf size (including rachis and petiole) with a leaf area meter ( LI 3100, LICOR, Lincoln, Nebraska, USA), lamina thickness with a micrometer (at three positions avoiding veins as much as possible) and leaf fresh mass. Wood specific gravity sampling followed the protocol described in Chave et al. (2006); all individuals were between 10 and 36 cm diameter at breast height (130 cm). Wood cores were collected from five individuals per species (the same individuals from which leaves were collected whenever possible), except I. acuminata for which samples from only three indi viduals were collected. Seeds were collected during March 2007, May June 2007, February July 2008, January August 2009 and May 2010 directly from multiple parent trees or from the ground near fruiting adults. Seeds were removed from pods and testa rinsed with de ionized water, towel dried and weighed for fresh mass. Leaves and seeds were dried at 60 C for three to four days and their dry mass determined, then ground for analysis of N with an elemental analyzer (Costech Analytical ECS 4010, Valenica, CA) a nd P with an ash digest ascorbate assay (Murphy


24 & Riley 1962). Seeds including paper thin seed coat for each parent tree were pooled as a replicate before grinding. Statistical Analysis The significant bias of individual distribution in relation to soil ty pes was tested with 2 test under the null hypothesis that equal numbers of individuals should grow on both soil types. Furthermore, species were ranked according to the percent of individuals found on residual soil. Changes in CII among species and life 2 test. For leaf functional traits, we examined the effects of soil preference (alluvial vs. residual), species nested within soil preference type and ontogenetic stage (sapling vs. adults) and interaction between ontogenetic stage and species nested within soil preference type. As sapling data were not collected for I. acuminata it was excluded from this analysis. The models were tested with and without the only species without soil bias, I. leiocalycina Seed and wood data were also tested for soil preference and species nested within soil conducted across all nine species. Results Species Distribution In 97 of the 104 survey locations at least one Inga individual > 50 cm tall was encountered. In total, 813 individuals > 50 cm tall were identified to species. We found 16 Inga species; the nine species with a total of at least ten individuals were used in f urther analyses (Table 2 1). Individuals of these nine species comprised 96% of all Inga individuals found in the survey. These nine species were classified according to their soil preference types based on the number of individuals occurring on residual a nd


25 alluvial soils. Eight species showed distribution al biases to either residual (5 spp) or alluvial (3 spp) soil, while one occurred on both soil types (Table 2 1). The CII 2 = 36.7 p = 2 = 79.3, p < 0.001). 2 = 75.3, p < 0.001). Functional Traits All of the leaf functional traits examined, except for leaf lamina thickness, were significantly different between soil type preference groups regardless of whether the only unbiased species ( I. leiocalycina ) was excluded from the analysis (Tables 2 2, 2 3) or included (results not shown). Residual specialists had higher values of LMA and leaf density than alluvial specialists, while the unbiased species ( I. leiocalycina ) had intermediate LMA, low lamina thickness and high leaf density as adults (Fig. 2 2 ). Nutrient concentrations in leaf laminas were higher for alluvial specialists than residual speci alists per unit mass (%N and %P; Table 2 2), but not per unit area (Table A 2). Residual specialists also had higher WSG than alluvial specialists, although there was a large overlap between the two groups (Table 2 2). LMA, lamina thick ness, and lamina density tended to be higher for adults than for saplings, although the magnitude of change observed between the two ontogenetic stages differed among species (Tables 2 2 2 3 ; Fig. 2 1 ). However, the degree of ontogenetic plasticity was un related to soil type preference, because there were no significant interactions between stag e and habitat preference type. The only exception was lamina N, which showed a marginally significant interaction of ontogenet ic stage with soil preference. Lamina %N did not differ significantly between saplings and adults (Table 2 2 ), but N per unit area was higher for adults than saplings due to greater LMA


26 in the former (Table A 2). Lamina P was higher for adults in only some species (Table 2 2 ). Seed mass also differed significantly among species, and residual specialists had greater seed mass than alluvial specialists (Tables 2 2 2 3). Seed N was significantly higher for residual specialists than alluvial specialists, but seed P did not differ among species. T he one unbiased species, I. leiocalycina had values intermediate between the average values for the two specialist groups. Discussion Habitat Association Our trail based survey demonstrated that distribution of eight of the nine most common species of Ing a exhibited significant edaphic biases, confirming the soil type associations observed by experts (Zamora & Pennington 2001). Inga species studied in other Neotropical forests also show non random distributions in relation to slope (Harms et al. 2001), soi l water content and pH (Endara & Jaramillo 2011) and soil nutrient availability and geographic distance (Dexter et al. 2010). Soil type preference of each species was not absolute; some individuals were found on their non preferred soil type, a pattern oft en observed in other studies of soil type preferences (e. g., Clark Clark & Read 1998, La Selva; Russo et al. 2005, Malaysia; Queenbrough et al. 2007, Ecuador). This is not surprising, as seeds can easily disperse to the other soil type. Stochasticity and additional dimensions in ecological niches, such as light availability, water availability and forest successional stage may promote establishment of juveniles on non preferred soil types (Baraloto et al 2007). After initial establishment, ecological sort ing continues throughout juvenile to adult transition, as found by Russo et al. (2005) in a Malaysian forest plot and Clark et al. (1998) in the same forest for non Inga


27 species. The current study focused on functional traits associated with soil type pref erence, and the study design would not allow us to examine temporal changes in species distribution patterns with ontogeny. Such data collected with appropriate spatial sampling would provide greater insights on the process of trait based habitat filtering Due to recent and rapid diversification, phylogenetic relationships within Inga are difficult to resolve (Dexter et al. 2010), and there was no obvious phylogenetic bias in soil preference type among the nine Inga species in the study (Fig. A 1). Perhap s, soil type preference is an evolutionarily labile character as found by Russo et al. (2007) for a broad range of Malaysian tropical tree species. Alternatively, trait diversification due to recent niche sorting within a speciose genus can show weak phylo genetic signals, as found for chemical defense traits of 37 Inga species in Panama and Ecuador (Kursar et al. 2009), and for Quercus spp. that distribute along soil fertility gradients (Cavendar Bares et al. 2004). Soil Type Preference a nd Functional Trai ts All traits of both adults and saplings, except for leaf lamina thickness, N per unit area, and P in seed, showed significant difference in relation to the soil type preference of the species (Tables 2 3, 2 4, A 2). For the eight species that exhibited a significant bias to one or the other soil type, a sufficient number of individuals could be sampled only in the preferred soil type. It is possible that plastic responses to soil nutrient availability may contribute to the observed differences among speci es, but results for three species sampled from both soil types suggest that phenotypic plastic responses to in situ soil types appeared small relative to inherent differences among species (see Table A 1). The relative contribution of genetic basis and pla sticity, however, differ among traits, and it is not possible to know whether adaptation or acclimation is


28 responsible for observed variations when plants are sampled only in their typical habitats (Endara & Coley 2011). For example, Fine et al. (2006) fou nd that habitat preference of species (to infertile white sand vs. richer clay soil), but not in situ soil type, affect leaf protein concentration, but the opposite was the case for leaf toughness. The results supported our a priori expectation that LMA s hould be higher for infertile residual soil specialists than alluvial soil specialists, a result also found for 79 species in Australia (Wright & Westoby 2003) and other studies that compared resource rich vs resource poor habitats (Reich Ellsworth & Uhl 1995; Wright et al. 2004; Poorter et al. 2008). Change in LMA may be achieved either by increasing lamina thickness, lamina density (dry mass per volume), or both (Onoda et al. 2011; Westbrook et al. 201 1 ). We found that only leaf density, but not lamina thickness, increased with specialization to less fertile soil. Parallel to this, across 19 Bolivian tree species, LMA and lamina density, but not lamina thickness, were associated with slow growth strategies of shade adapted species (Kitajima & Poorter 201 0). Thus, lamina density, rather than lamina thickness, is the general correlate of slow growth syndromes in resource poor habitats, just as high WSG shows general association with slow growth of low resource specialists across a broad range of species (Po orter et al. 2008; Chave et al. 2009 ; Poorter et al. 2009; ). This is a broadly convergent pattern despite significant phylogenetic signals across broad phylogenetic ranges for both WSG and lamina densi ty (Chave et al. 2009; Westbrook et al. 2011). The dive rgence in these traits within a single species rich genus in relation to soil type preference strongly suggests the adaptive significance of high tissue density in leaves and stems as part of the adaptive trait syndromes to resource poor environments.


29 Lea f N was significantly lower for residual soil specialists than alluvial soil specialists (adult leaf mean %N = 3.29 and 3.79%, respectively, Table 2 2 ), although there was no significant difference for N per unit lamina area (Table A 2), which suggests tha t leaf N reflects allocation strategies for optimal light and nitrogen use (Hikosaka & Hirose 2000). The observed %N values were high relative to other tropical trees in Fabaceace (mean N = 2.5%, Townsend et al. 2007). Similarly, leaf P concentrations (adu lt leaf mean = 0.13 and 0.19%, for residual and alluvial soil specialists, respectively; Table 2 2 ) were high compared to other tropical species (mean = 0.09%; Townsend et al. 2007). Inga species at LS had high values of leaf N and P, possibly because even residual soil at LS is relatively fertile compared to many other tropical sites (Porder et al. 2006). But, leaf N concentrations were high even in comparison to other legume species at LS (e.g., Pentaclethra macroloba ; Porder et al. 2006).We recognize tha t ecological niche dimensions other than soil fertility may also influence functional trait evolution. For example, of the five residual soil specialists in our study, I. acuminata had the highest WSG (Table 2 2 ) and the lowest LMA (Figure 2 1 A ). I. acumin ata is also the only species in the group classified as a subcanopy tree and thus other variables, such as light availability, may contribute to the observed differences in trait values. Ontogenetic Changes in Leaf Traits All traits examined in this study differed between saplings and adults, but species varied widely in the degree of ontogenetic change within, rather than between, soil type preference groups (Table 2 2 ). These changes may reflect changes in height per se or changes in microenvironmental fa ctors with height growth. Crown illumination index was higher for adults than for saplings, but hydraulic limitation associated with tree height


30 might also be important in explaining height associated variati on in LMA (Thomas & Winner 2002; Cavaleri et al. 2010). Both components of LMA, lamina thickness and lamina density, increased from saplings to adults (Fig. 2 1), although such increases in lamina thickness may not be universal (Kitajima & Poorter 2010). Leaf N concentration did not differ between sapli ngs and adults, but adults had greater N per unit area, which is an adaptive plasticity response to increased light availability experienced by adult leaves. C rown illumination index was not different among species at the sapling stage, however it was at t he adult stage (data not shown). The species differences in degrees of CII change from saplings to adults may be responsible for species differences in degrees of ontogenetic changes in leaf traits. Seed Traits Particularly interesting among our results wa s seed N, which was higher for infertile residual soil specialists. This was the opposite of the trend for leaves, and also the opposite of the trend that would be expected if seed N reserves merely reflect soil N availability. Grubb & Coomes (1997) found that tall trees in the infertile soils of caatinga forests have seeds with higher N compared to tree species occurring in adjacent more fertile soils. But our results differed from Grubb & C o omes (1997) for seed size; seed size tended to be greater for Ing a spp. that prefer less fertile soil, suggesting that both higher nutrient concentrations and larger seeds may be selected in infertile soil, because the larger N capital is advantageous for seed reserves to support seedling nutri ent demands (Kitajima 2002 ; Groom & Lamont 2010). While the advantage of seed size per se may be equivocal in adaptation to infertile soil (Leishman et al. 2000; Russo et al. 2007), higher total N in seeds may be selected for in infertile soil as a strategy to prolong the duration of seed reserve dependency necessary for initial seedling


31 establishment (Kitajima 2002). Even though Inga seedlings form N fixing nodules and the two soil types did not differ in total soil N, higher initial N availability from seeds may be adaptive for in itial root development and establishment of mycorrhizal association. Following the same logic, we hypothesized that seed P concentration should be higher for residual soil specialists, but this expectation was not supported (Table 2 2 ). It may be that, reg ardless of soil fertility, the optimal seed P concentration selected for must be high enough to meet the seedling P demands until the time or ontogenetic stage when seedlings establish symbiotic associations with mycorrhizal fungi (Janos 1980). Summary In ga is one of the hyper diverse tropical tree genera that have gone through rapid and recent diversification. Eight of the nine common Inga species in this lowland tropical wet forest exhibited clear preference to either rich alluvial soil or less fertile r esidual soil of volcanic origin. The patterns of leaf functional traits of adults and juveniles, as well as wood specific gravity, were consistent with the expected trait differences as a result of habitat filtering in resource rich vs. less rich environme nts. Species that become successfully recruited as adults on less fertile soils have high LMA, high tissue densities of leaves and wood, and low N and P per unit mass in leaves, observations that are typically associated with slow growth and high survival as a life history strategy. In addition, N concentrations in seeds were higher for residual specialists. This suggests that nutrient allocation strategy to seeds also reflect selection for different seedling establishment strategies in fertile vs. less fer tile soils. We conclude that community assembly is likely to reflect trait mediated niche s orting at multiple life stages.


32 Table 2 1. Distribution of Inga species along 104 50 m long trail segments at La Selva Biological Station for which at least a tota l of 10 individuals taller than 50 cm 2 and p for soil type preference for residual soil, alluvial soil or without significant bias. The species were ranked from greater to lesser degre e of bias to the residual soil based on the percent of individuals found on residual soils. Species Rank No. on residual soil No. on alluvial soil Soil type p reference 2 p I cocleensis 1 11 1 Residual 14.8 < 0.001 I. acuminata 2 10 1 Residual 13.2 < 0 .001 I. thibaudiana 3 117 14 Residual 154.0 < 0.001 I. alba 4 20 3 Residual 25.1 < 0.001 I. pezizifera 5 70 23 Residual 59.6 < 0.001 I. leiocalycina 6 25 37 none 0.2 0.25 I. oerstediana 7 21 139 Alluvial 41.5 < 0.001 I. sapindiodes 8 9 192 Allu vial 95.1 < 0.001 I. marginata 9 3 73 Alluvial 37.1 < 0.001


33 Table 2 2 Species means for seed mass, N and P concentrations, and wood specific gravity (WSG) of nine Inga species (See Table 1 for Species identity, Table 3 for ANOVA results). Also shown a t the bottom are means for soil type preference to residu al (R) and alluvial (A) soils. For leaf traits, n = 10 individuals per species except n = 7 for Sp. 2. For WSG, which was determined only for adult trees, n = 5 except n = 3 for Sp. 2. Letters that f ollow mean values indicate the results of post hoc Tukey Kramer pairwise comparisons. ND = no data. Species rank Soil Seed mass (g) N (%) P (%) WSG (g cm 3 ) Seed Sapling Adult Seed Sapling Adult Adult 1 R ND ND 3.23 d 2.77 d ND 0.11 de 0.10 d 0. 59 a 2 R 0.30 bcd 3.67 bc ND 3.53 b 0.15 ND 0.14 c 0.61 a 3 R 0.13 d 3.31 bcde 3.13 d 3.06 cd 0.19 0.10 e 0.11 d 0.44 b 4 R 0.30 bcd 2.03 de 3.34 cd 3.73 b 0.14 0.11 de 0.16 bc 0.52 ab 5 R 0.73 a 4.99 a 3.67 b 3.44 b 0.20 0.14 cd 0.14 c 0.44 b 6 N 0.71 a 3.38 bcde 3.66 b 3.43 bc 0.19 0.15 c 0.15 c 0.53 ab 7 A 0.27 c 3.26 bd 3.54 bc 3.56 b 0.18 0.15 bc 0.17 bc 0.47 ab 8 A 0.36 b 2.94 bcde 4.34 a 4.28 a 0.18 0.19 ab 0.19 ab 0.49 ab 9 A 0.15 d 2.48 ce 3.19 d 3.53 b 0.16 0.20 a 0.22 a 0.41 b Soil pref. R 0.49 4.03 3.34 3.29 0.18 0.12 0.13 0.51 means A 0.30 2.90 3.69 3.79 0.17 0.18 0.19 0.46


34 Table 2 3 The results of nested 2 way ANOVA testing the effects of soil preference (alluvial or residual), ontogenetic stage (sapling or adul t), species (Sp, nested within soil preference type), and their interactions on five leaf tr aits. LMA =leaf mass per area. Shown are p values with degree of freedom (df) at the top. Leaf trait analyses excluded I. leiocalycina (with no soil preference) and I. acuminata (with no sapling data), and seed trait analyses excluded I. leiocalycina and I. cocleensis Soil pref Stage Sp(soil pref) Stage*Sp(soil pref) Stage*soil pref df 1 1 5 5 1 Leaf traits (determine for saplings and adults) LMA < 0.00 1 < 0.0 0 1 < 0.0 01 0.00 3 0.06 Leaf thickness 0.16 < 0. 001 < 0. 001 < 0.0 01 0.0 9 Leaf density < 0.0 01 < 0. 001 < 0. 001 0.0 01 0.72 N (%) < 0.001 0.87 < 0.001 < 0.001 0.014 P ( % ) < 0.001 0.011 < 0.001 0.0004 0.75 Seed traits and wood specific gravity (WSG) Seed N (%) 0.002 < 0.001 Seed P (%) 0.95 0.10 Seed mass < 0.001 < 0.001 WSG 0.001 0.001


35 Figure 2 1. Three leaf traits (mean + s.e.) of saplings (left side) and adults (right side) of nine Inga species, listed in the order of strong to lesser bias to residual soil (Tables 1, 2). A) Leaf mass per area (LMA, g m 2), B) lamina thickness (mm) and C) lamina tissue density (g cm 3). Open bars for residual soil specialists, dotted bars for unbiased species, and hatched bars for the alluvial so il specialists. No data available for sapling leaves of Species 2.


36 CHAPTER 3 SEED AND SEEDLING TR AIT COVARIATIONS AMO NG SPECIES IN INGEAE (FABACEAE) FROM TWO NEOTROPICAL FORESTS Background Seed and seedling traits are of interest to ecologists because the y should vary in relation to seedling recruitment strategies (Grubb 1977; Hanley et al. 2004; Leck, Simpson & Parker 2008). Because seedling growth and development depend on e nergy (carbohydrates and lipids) and mineral nutrients stored in seeds ( Fenner & Thompson 2005), size and composition of seed reserves should be subject to natural selection. Indeed, plant species differ in size and relative concentrations of resources in seeds, as well as manners in which seed derived resources are utilized by develop ing seedlings (Kitajima 1996). Among seed traits, seed size (dry mass, volume or length of the seed) is perhaps the most widely studied ( Moles & Westoby 2004). It is widely recognized as a key trait involved in life history trade offs, exhibiting interspec ific correlations with fecundity ( seed number per plant per year) time to reach reproductive maturity, size and longevity of reproductive adults and dispersal mode (Moles & Leishman 2008). Furthermore, large seed size is known to be associated with large initial seedling size (Moles & Westoby 2006) storage type cotyledons (oppos ed to photosynthetic cotyledons; Kitajima 1996; Ibarra Manriquez Ramos & Oyama 2001) and reduction in initial seed reserve transfer to the seeding (Green & Juniper 2004) Optimal seed size also appears to vary with regeneration habitats. Species whose seedlings establish in forest shade tend to have large seeds (Leishman et al. 2000), because of the likely advantage of either large initial seedling size where growth rates are const rained by low demands (Kitajima 1996). Although large seeds may also provide an advantage in


37 environments where infertile soils constrain growth rates, the relationship of seed size with soil fertility is equivocal ( Fenner & Kitajima 1999 ). Small seedlings without a well developed root system and/or symbiotic soil microbes must obviously depend on mineral nutrients stored in seeds. Information on how seed nutrient concentrat ions vary with seed mass will contribute to a better understanding of seed nutrient reserve size as part of seed and seedling trait syndromes. Although less widely studied than seed mass, species exhibit substantial variation in concentrations of mineral n utrients stored in seeds, including concentrations of the two macro nutrients, nitrogen ( N ) and phosphorus ( P ) (Barclay & Earle 1974; Fe nner & Lee 1989; Kitajima 2002). Contrasting selective forces may influence how concentrations of non structural carbohy drates, lipids and mineral nutrients are related to seed size (Kitajima 1996). For instance, small seed size may be compensated by high nutrient concentration in seeds. On the other hand, when the availability of a particular mineral nutrient is limited in the habitat, both larger seed size and high nutrient concentrations might be selected to enhance the total nutrient pool size in seeds. However, seed size (Moles & Westoby 2004) and nutrient concentrations (Lord, Westoby & Leishman 1995) exhibit significa nt phylogenetic signals. Hence, any observed correlations or a lack of an expected correlation may be due to phylogenetic constraints. Thus, comparison among related taxa may be more informative in exploring the evolutionary implication of variations in se ed size and nutrient concentrations than comparison across multiple unrelated taxa. Nutrient concentrations in seeds may also differ in relation to species differences in seedling tissue nutrient concentrations and intrinsic growth rates. Compared to


38 inher ently slow growing species, fast growing species have higher N and P concentrations in leaves and roots (Lambers et al. 2008), and allocate proportionally more biomass to leaves than to stems and roots ( i.e., lower root:shoot ratio, higher leaf area ratio ) Thus, it may be reasonable to expect that inherently fast growing species should have higher seed N and P concentrations to facilitate high optimal concentrations of N and P in the seedling. However, few empirical studies have examined whether concentrat ions of N and P in seedling organs are correlated with seed N and P concentrations, or how seed N and P reserves are allocated to leaves, stem and roots of seedlings during the initial seedling development. In Chapter 2, we compared nine Inga species that contrast in habitat preference (to fertile alluvial soil vs. less fertile residual soil), and found that seed N concentration was lower for alluvial soil specialists than for residual soil specialists, even though adult and seedling leaves of the former ha d higher N and P concentrations. Thus, N and P concentrations in seeds may be decoupled from the selection for optimal N and P concentrations in adult and sapling leaves, because natural selection favors increased maternal support of seedling nutrient dema nds in infertile soils. In the current study we examined allocation patterns of N, P and biomass in seeds and seedlings for species within a hyper speciose genus Inga and four additional genera within the tribe Ingeae (Fabaceae). Fabaceae species, includi ng those in Ingeae, often form symbiotic relationships with nitrogen fixing bacteria, which is considered to be important for meeting their high N requirements (Mckey 1994). Seedling traits were compared at the first leaf expansion stage, using seedlings g rown from seeds under deep shade in a shade house. This standardization of ontogeny and


39 environment allowed us to examine allocation of seed derived resources rather than autotrophically acquired resources, because seedling development largely depends on s eed reserves until the completion of the first leaf expansion in shade (Kitajima 2002). During this heterotrophic phase of growth and development, it is expected that respiration for growth and maintenance should result in a net loss of biomass, while nutr ient leaching may result in loss of nutrient pool size (Kitajima 1996). We asked the following questions: (1) How do size (dry mass), N and P contents of seeds and seedlings co vary across related taxa within the tribe Ingeae? (2) Are N and P differentiall y allocated from seed to seedling organs (leaves, stems or roots)? (3) What are the proportions of mass, N and P reserves in the seed retained in individual seedlings? Methods Study Sites We sampled Ingeae species from two Neotropical forest sites. The fi rst was Barro Colorado Nat ure Monument (BCNM) (9 situated in the Panama Canal This site is mostly covered by tropical moist forests of various successional ages ranging from young secondary forests to old growth stands T he second site is a tropical wet forest that also ranges in successional age from second growth to old growth stands within La Selva Biological Station (LS) (10 84 in the central lowlands of Costa Rica located adjacent to Br aulio Carrillo National Park. BCNM has a marked dry season with <100 mm per month from January to May, while LS has no marked dry season. Soils at both sites are clay rich oxisol s although additional soil types can be distinguished within each site B rown fine loam and red light clay are distinguished at BCNM (Die trich, Windsor & Dunne 1996)


40 whereas old alluvium and residual soils are distinguished at LS (Sollins et al. 1994). Despite differences in annual rainfall and within site variations overall soil nutrient availability is considered to be similar between BCNM and LS relative to the wide range of soil fertility observed across Neotropical forests (Powers, Treseder & Lerdau 2005). Study Genera We sampled a total of 23 species from the two study sites Seventeen species belonged to genus Inga which is the most speciose genus in Ingeae, while the remaining six species belonged to four other genera within Ingeae. BCNM and LS each have approximately 20 species of Inga with some overlap between the two si tes Only one species, however, could be sampled from both sites in the current study due to differences in abundance and seed availability between the two sites. Rapid and recent speciation within Inga (Richardson et al 2001) allowed us to examine potent ial correlations among resource allocation traits of seeds and seedlings among closely related species with a common phylogenetic background. Like many other tropical genera Inga species are known to exhibit soil type association (Harms et al. 2001; Dexter Pennington & Cunningham 2010 ; Chapter 2 ). The maximum adult height of Inga species range s from 12 m to 40 m (Zamora & Pennington 2001). We sampled two species in each of the gen era Cojoba and Zygia a t LS The other two genera, Abarema and Balizia were b oth repres ented by only one species each at LS. Balizia is a canopy emergent species with maximum height >35 m (Finegan, Camacho & Zamora 1999). Th e seed coat was very thin and weight was negligible in all species except for Balizia and Abarema All specie s except for Balizia had recalcitrant seeds and thus seeds were kept moist and planted within 24 hours of collection if they were to be germinated


41 The only exception, Balizia had thick and hard seed coat that had to be scarified mechanically by sanding to break physical dormancy to stimulate germination. BCNM Study Seeds from six species of Inga wer e collected at BCNM from March through April 2006. At least ten seeds of each species were dried at 60 C for seed trait measurements. Most seeds were planted in plastic trays containing a moist mix consisting of equal parts of vermiculite and sand, which were expected to a provide negligible amount of N Seeds were germinated in a shade house at ~2% f ull s unlight, using a combination of neutral shade cloths and a wave length altering plastic filter (Lee et al. 1997) to simulate the light in the natural forest understory. This light level is similar to that of the forest understory (Chazdon & Fetcher 1984) Seedlings were allowed to grow until their first leaves were green and fully expanded. Th e first leaf stage was chosen because it is an easily identifiable and comparable developmental stage (Green & Juniper 2004). Seedlings were then harvested, separated into leaves, stems, cotyledons and roots, and leaf area was measured. Seeds and seedlings were dried at 60 C. These data were used to calculate allocation patterns such as root to shoot ratio (RS, root ma s s /(stem + l eaf mass) ) and leaf mass ratio (LMR lea f mass/ whole plant mass ). Seed and seedling organs were also analyzed for N with an elemental analyzer (Costech Analytical ECS 4010, Valenica, CA) LS Study Seeds from 18 species of trees in the tribe Ingeae (including 12 Inga species) were collected at LS from March to May 2007, February to August 2008, and January to August 2009 and May 2010 All seeds were rinsed with deionized water. After determining fresh mass within several hours of collection, one half of seeds collected


42 from each mother tree were d concentrations The remaining seeds were germinated to collect seedling data. The fresh and dry seed mass data were used to quantify the relationship between wet and dry mass of seed for each par ent tree. The linear r egression equation for each mother tree was used to estimate the dry mass of each seed planted to collect seedling data. The seed germination conditions were similar to the BCNM study, except light intensity was slightly higher (<3%) and 100% river sand wa s used for seeds planted in 2008 and 2009. Sand was cleaned in a 10 % bleach solution and rinsed with water and expected to provide negligible amount of P Seedlings were grown until the first leaf became green and fully expanded (typically 3 5 weeks ; Tabl e 3 1 ). Two species ( B. elegans and A. adenophora ) had seed coats that could not be easily removed from the cotyledons and embryo. For these species, the seed coats that were naturally shed during germination were collected and mass and N content of seed c oats were subtracted from the seed data. At harvest seedlings were separated into leaves, stems, roots and cotyledons, and leaf area was measured Cotyledons of all species except B. elegans were still attached at harvest, though a few individuals of othe r species had lost one cotyledon, and only those cotyledons that were still attached were analyzed. Then all organ We used b iomass data to calculate RS, LMR, and the ratio of seed mass to seedling mass Seeds and seedlings were first p ooled by parent tree and then by date of collection for nutrient analysis which required a minimum of 100 mg of dry ground sample. N was analyzed as described for BCNM, and P was analyzed using a dry ash ammonium molybdate assay (Murphy & Riley 1962). Bec ause P analysis i s less reliable when less than 50 mg of sample is used, not all species could


43 be analyzed for P due to insufficient sample availability We used the nutrient data to calculate proportion of transfer of N and P reserves from seed to seedlin g ( seedling N or P pool / seed N or P pool ) Statistical Analysis S pecies mean trait values were used for interspecific correlation analyses. When there was only one mother tree, the mean of seed or seedling traits for that tree were used. N on parametric te sts ( Spearman rank correlation and Kruskal Wallis test ) were used to analyze most of the data because standard transformation, such as log transformation, could not achieve normality in many variables N utrient concentration s between organ types were teste d with paired t tests. Results Two species, B. elegans and A. adenophora had smaller seeds and differed in cotyledon morphology compared to the other Ingeae species in this study. Both species ha d small photosynthetic storage cotyledons that were relative ly short lived, and seed coats were shed during germination. The remaining species in the study had thick storage cotyledons that were at the ground or raised somewhat above the ground. A threefold variation in seed mass was found within the genus Inga wh ich was smaller than the variation observed within the tribe Ingeae (Table 3 1). The magnitude of interspecific variations were less for seed N and P than for seed mass; seed N concentration varied from 20 63 mg/g (Table 3 1) and seed P concentration varie d 1 3 mg/g (Table 3 2). Seed N and P concentrations were correlated with each other (Table 3 3), but neither was correlated with seed mass (Fig 3 1 B 1 ). N and P concentrations in seedling leaves, stems and roots varied significantly among species (Table B 1), so did biomass allocation patterns (RS and LMR).


44 However, none of these traits were correlated with seed mass (Table 3 3). Seedling leaf N concentration was high (ranging from 40 90 mg/g) (Table 3 1). Similarly, N concentrations in stem and root, as well as P concentrations in all organs (measured in LS species only), were high (Table 3 1 3 2). In the majority of species, leaves had higher N concentrations compared to stems and roots, which had N concentrations similar to each other (Fig. 3 2 B 2 ). N concentrations in stems, roots and cotyledons, but not in leaves, were positively correlated with seed N (Table 3 3; Fig 3 2), however the correlations of stem and root N concentration with seed N concentration were non significant when B. elegans and A. adenophora were excluded from the analysis. P hosphorus concentration in stem, but not in leaves, roots or cotyledons, was positively correlated wi th seed P concentration (Table 3 3). P hosphorus concentrations in leaves, stems and roots were not correla ted with each other (Table 3 3). Interestingly seedling leaf N and P concentration were uncorrelated with N and P concentrations in adult leaves (Table 3 3). The total biomass, as well as pool size of N and P, decreased during the ontogenetic transition f rom seed to the first leaf stage. The proportion of seed N and P retained in seedlings was not different among species (Fig 3 3). The ratio of seedling mass (excluding cotyledons) to seed mass was different among species and ranged from 34% to 61% (Fig 3 3) and it was negatively correlated with seed mass (Table 3 3), indicating that small seeded species used a larger proportion of biomass during the initial development through first leaf expansion compared to large seeded species (Fig 3 3).


45 Discussion We asked three questions about seed and seedling traits at the end of the introduction, for each of which the following results were found. (1) N and P concentration in seed and seedling organs differ significantly, but independently of seed size, among In geae species. (2) N and P concentrations in seedling leaves were uncorrelated with N and P concentrations in seed, stem, and roots, whereas the N correlations were correlated among seed, stem and roots. (3) The p roportion of N and P pools retained from see ds to first leaf stage seedlings was higher than the proportion of biomass retained; the former was independent of seed size, while the latter was negatively correlated with seed mass. Below, we discuss our findings and what may be the explanations for the se observed patterns. Ingeae species in this study exhibited significant interspecific variation in seed size (from 0.02 g to 1.8 g), although this represents a small range compared to the 10 11 fold variations observed among the Angiosperms and 10 5 fold va riations often observed among tropical trees within a forest community (Moles et al. 2005; Kitajima 2007). Not surprising for a species rich family, Fabaceae exhibit the largest range of seed sizes among Angiosperm families, and all seed sizes in this stud y were larger than the mode of seed size for the family (Corby Smith & Sprent 2011). Seed size is known to be phylogenetically conserved (Lord, Westoby & Leishman 1995; Moles et al. 2005), and indeed seed size variation observed within the genus Inga was much less than across the tribe Ingeae (Table 3 1). The available literature suggests no consistent relationships between seed mass and nutrient concentrations. Comparing species ranging broadly in their phylogenetic backgrounds, Grubb et al. (1998) found seed nutrient concentration to be negatively correlated with seed mass while Soriano et al


46 (2011) did not. However, Corby et al (2011) found that among legumes seed N concentration is negatively correlated with seed mass because seed mass increases faste r than N concentration. Leaves, stems and roots of the young seedlings in this study exhibited much higher tissue N concentrations than seeds or adult leaves of the same species (Table 3 1; Chapter 2). The seed contained elevated concentrations of nutrient s, and nutrient concentration per unit dry mass apparently increased during the initial seedling development because nutrient loss was proportionally less than biomass loss (Fig 3 3). High seedling nutrient concentrations are believed to be important for legume seedlings because establishing symbiotic relationships with N fixers requires high initial N and P investment (Pate 1986; McKey 1994). Ingeae seeds are arrested embryos that store most of their resources in storage cotyledons, and their germination morphologies resemble those of common beans ( Phaseolus spp.) and peas ( Pisum spp.). During the seed reserve dependent metamorphosis to the first leaf stage seedlings, there was a net loss of biomass (Fig. 3 3A), most likely because carbohydrates and lipids were expended for growth, transport and maintenance respiration. The pool size of N and P in the seedling excluding cotyledons represents the total of N and P that had been transported from seed cotyledons to the stem, leaves and roots (Fig. 3 3B, C). Exc ept for B. elegans cotyledons were still attached to the seedling at harvest, and cotyledons still contained substantial quantity of mass, N and P reserves even though the majority of reserves had been exported out of cotyledons within 23 36 days after ge rmination. Thus, seedlings were at least partially reliant on maternal reserves, which may be particularly important when they have to recover from damage or establish relationships


47 with symbionts (mycorhizzae, rhizobia). The elevated concentrations of N a nd P in seedling stems, which were correlated with seed N and P concentrations, may continue and their importance should decrease as seedlings start to acquire N and P from the soil and microbial symbiosis. Likewise, the elevated concentration of N and P in seedling leaves and roots may also be temporary. The situation may be analogous to luxury consumption (Chapin 1980); it may be advantageous for seedlings to store N and P in nonphotosynthetic organs when they have access to more n utrients than they can use (Lawre nce 2003). The results also suggest that N and P concentrations in seedling leaves reflect the optimal N and P allocation strategy. A high proportion (> 40%) of seed N and P pools transferred to seedlings w ere allocated to leaves. This resulted in leaf N of seedlings approximately 10 mg/g higher than adults (26 to 43 mg/g) of the same species (seedling leaf N reported in Table 3 1 compared to adult leaf N reported in Chapter 2). Mckey (1994) hypothesized tha t legume seedlings have high N concentrations to support high photosynthetic rates which should lead to enhanced growth of seedlings. Moreover, the N concentration of seedling leaves were in the same range as those of older tropical legume seedlings grown with fertilizer in a greenhouse (Huante, Rincon & Acosta 1995). Thus, it seems that seed reserve alone was sufficient to achieve such high leaf N concentration in the first seedling leaf, even though N availability was negligible from the soil medium used in our study. Additionally, the lack of correlation between seedling and adult leaf nutrient concentrations in all species, may hint at large ontogenetic shifts in leaf nutrient concentrations that should occur over time between seedl ing s and adults


48 The proportion of seed biomass committed to permanent seedling organs at the first leaf stage decreased with increasing seed mass (Table 3 3 ; Fig. 3 3A ), because cotyledons represented greater proportion of seedling biomass with increasing seed size. This resu lt was in agreement with the findings of Green & Juniper (2004) for 15 Australian rainforest species, reporting the proportion of seed reserves kept aside as storage increases with seed size. It was not clear, however, what proportion of cotyledon mass rem aining may represent usable resources such as lipids, starch and protein, opposed to non mobile mass. Alternatively, species differences in inherent growth or metabolic rates may explain differences in net biomass loss. However, biomass allocation traits t hat are often associated with relative growth rates, such as RS and LMR, were not correlated with seed mass, similar to findings of Jurado & Westoby (1992). On the other hand, the proportion of seed N and P transferred from cotyledons to seedling stem, lea ves and roots did not differ among species, which may mean that the optimal strategy is to retain seed nutrients regardless of the seed size. Summary This study explored the relationships between seed reserves and seedling allocation of those reserves in t he legume tribe Ingeae. Fabaceae are known for their species richness and functional diversity, but because we purposefully sampled species within Ing e ae, the majority of the species we sampled exhibited similar functional morphology of cotyledons and deve lopmental patterns and this may be why few significant correlations were detectable. Nevertheless, we found positive correlations between seed N and P concentrations, and N and P concentrations between seed and stem. The data from this study add to a growi ng collection of data on plant functional traits and demonstrate that certain general patterns in functional trait values, such as


49 negative correlation between seed size and seed N concentrations, may not be detectable when analyses are confined to closely related species.


50 Table 3 1. Species mean values of seed mass (g) excluding seed coat, seedling mass (excluding cotyledon) (g) and nitrogen (N) per unit dry mass (mg/g) in seeds and seedling organ s. Species are listed in order of increasing seed mass; six species sampled from BCNM are indicated with asterisks (*), while the r emaining were sampled from LS. Age is the number of days between germination and seedling harvest. The sample size indicates the number of mother trees from which seeds were collected, followed by the total number of seeds in parentheses Species Mass (g) Age Sample N (mg/g) Seed Seedling (day) Size Seed Leaf Stem Root Balizia elegans 0.023 0.013 23 1 ( 42 ) 54.6 81.0 85.4 52.1 Abarema adenophora 0.057 0.0 34 17 2 ( 14 ) 62.6 89.1 84.4 52.4 Inga thibaudiana 0.130 0.0 57 33 2 ( 1 2) 33.3 40.5 44.8 43.3 Inga cocleensis* 0.143 0.0 47 33 2 ( 9 ) 23.9 52.5 37.6 33.9 Inga marginata 0.147 0.0 64 22 5 ( 33 ) 24.8 40.3 35.7 34.1 Cojoba valerioi 0.221 1 (16) 28.6 Inga sapindoides* 0.237 0.1 20 33 3 ( 10 ) 26.7 57.1 39.6 36.7 Inga acuminata 0.246 1 (5) 36.7 Inga oerstediana 0.269 0.1 19 26 4 ( 19 ) 32.6 51.5 41.0 35.5 Inga laurina* 0.277 0.1 31 30 1 ( 10 ) 27.9 46.0 40.9 40.6 Inga multijuga* 0.286 0. 133 25 2 ( 11 ) 28.2 50.2 42.6 38.6 Cojoba catena ta 0.363 0. 135 32 7 ( 109 ) 22.4 42.2 34.9 37.5 Inga sapindoides 0.451 0. 202 34 5 ( 78 ) 30.5 55.2 33.4 35.5 Inga ruiziana 0.382 1 ( 13 ) 29.4 Inga goldmanii* 0.392 0. 143 32 2 ( 10 ) 20.4 43.5 34.2 31.6 Inga samanensis 0.435 1( 16 ) 33.2 Zygia longifo lia 0.425 0. 131 24 5 ( 110 ) 20.5 48.1 35.5 31.4 Inga umbellifera* 0.463 0. 140 31 1 ( 10 ) 27.2 47.8 35.5 36.3 Inga tonduzii 0.641 1 (19) 32.6 Inga leiocalycina 0.710 2 (12) 33.8 Inga pezizifera 0.734 4 (24) 49.9 Inga jinicuil 0.777 2 (13 ) 45.5 Inga mortoniana 0.853 1 (12) 33.7 Zygia gigantifoliola 1.841 2 (19) 19.6


51 Table 3 2 Mean phosphorus (P) concentrations (mg/g dry mass) in seeds and seedling organs and relative growth rate (RGR) of seedlings for species sampled at LS. Analysis was done with seed excluding seed coats except for B. elegans and A. adenophora for which seed mass include d the seed coat that was difficult to separate seeds Species are listed in order of increasing seed mass. Species P (mg/g) Seedling Seed Leaf Stem Root RGR (g/g/day) Balizia elegans 2.8 5.1 6.1 6.3 0.025 Abarema adenophora 2.9 5.1 6.6 4.4 0.013 Inga thibaudiana 2 2.3 5.4 3.5 0.009 Inga marginata 1.6 2.9 2.4 2.5 0.019 Cojoba valerioi 1.9 Inga acuminata 1.5 Inga oer stediana 1.8 3.8 3.1 2.4 0.014 Cojoba catenata 1.6 3.4 2.2 2.7 0.010 Inga sapindoides 1.7 2.8 2.1 2.1 0.010 Inga ruiziana 1.5 Inga samanesis 2.0 Zygia longifolia 1.8 4.3 3.1 3.7 0.012 Inga tonduzii 1.9 Inga leiocalycina 1.7 Inga pezizifera 2.0 Inga jinicuil 1.9 Inga mortoniana 1.3 Zygia gigantifoliola 1.4


52 Table 3 3 Species mean values for cotyledon mass (g), N concentration (mg/g), P concentration (mg/g) and the number of cotyledons attached to the main seed ling axis at harvest. Data were collected from cotyledons that were still attached to the main seedling axis at the first leaf stage. Species Mass (g) N (mg/g) P (mg/g) number Abarema adenophora 0.011 44.9 1.5 2 Inga thibaudiana 0.038 38.4 1.1 2 Inga co cleensis* 0.034 24.8 2 Inga marginata 0.034 21.3 0.9 1.9 Inga sapindoides* 0.068 27.5 2 Inga oerstediana 0.077 37.1 1.0 2 Inga laurina* 0.042 25.3 1.9 Inga multijuga* 0.075 33.3 2 Cojoba catenata 0.128 19.0 1.2 2 Inga sapindoides 0.109 29.8 1.4 2 Inga goldmanii* 0.095 20.9 2 Zygia longifolia 0.168 16.5 1.2 2 Inga umbellifera* 0.169 29.1 2


53 Table 3 4 Spearman rank correlation coefficients ) for correlations between pairs of seed, seedling and adult traits using the combined data sets from both sites. Trait Trait p df Seed mass Seed N 0.09 0.68 22 Seed mass Seed P 0.42 0.08 16 Seed N Seed P 0. 55 0.019 16 Seed mass RS 0.22 0.43 12 Seed mass LMR 0.30 0.30 12 Seed mass RGR 0.45 0.26 6 Seed mass Seedling to seed N ratio 0.24 0.57 6 Seed mass Seedling to seed P ratio 0.71 0.11 4 Seed mass Seedling to seed mass ratio 0.71 0.047 6 Seed N Seedling leaf N 0.48 0.08 12 Seed N Seedling stem N 0.73 0.0032 12 Seed N Seedling root N 0.84 0.0002 12 Seed N Seedling cotyledon N 0.9560 <0.001 12 Seed P Seedling leaf P 0.18 0.70 5 Seed P Seedling stem P 0.93 0.0025 5 Seed P Seedling roo t P 0.5 0.25 5 Seed P Seedling cotyledon P 0.4286 0.3374 5 Seedling leaf N Seedling stem N 0.41 0.14 12 Seedling leaf N Seedling root N 0.30 0.30 12 Seedling stem N Seedling root N 0.70 0.0056 12 Seedling leaf P Seedling stem P 0.21 0.64 5 Seedling leaf P Seedling root P 0.32 0.48 5 Seedling stem P Seedling root P 0.68 0.09 5 Seedling leaf N Adult leaf N 0.07 0.88 5 Seedling leaf P Adult leaf P 0.32 0.48 5 Indicates statistically significant results


54 Figure 3 1. Species mean (A) seed N con centration (mg /g ) and (B) P concentration (mg /g ) plotted against seed mass. C losed symbols are Inga species and open circles are species in the tribe Ingeae outside of the genus Inga Lines are linear regression lines (A. R 2 =0.05 and B. R 2 =0.24)


55 Figu re 3 2 Species mean (A) N and (B) P concentration (mg/g dry mass) in seedling organs plotted against N and P concentration in seed excluding seed coat The three organ types are leaves (closed), stem (open), and roots (grey). The lines are regression line s for the organ types: leaves (black), stem (dashed) and roots (grey).


56 Figure 3 3 Ratio of (A) the seed ing mass to seed mass, and ratio of total seedling nutrient pool size to seed nutrient pool size of (B) N and (C) P, at the first fully expanded lea f stage for species sampled at LS Cotyledons of Balizia elegans ( BAEL ) were dropped before harvest and were not included here. Phosphorus data were not available for BAEL and Abarema adenophora ( ABAD ) due to small seed size and limited sample availability Species names were abbreviated : Inga thib au diana (INTH), I. marginata (INMA), I. oerstediana (INOE), I. sapindiodes (INSA), Cojoba catenata (COCA) Error bars are standard error.


57 CHAPTER 4 EFFECTS ON SEED RESERVE AND SOIL NIT ROGEN ON EARLY SEEDL ING GROWTH OF BALIZIA ELEGANS Background Most tropical trees start life as seedlings in the shaded understory, depending on seed reserves for initial growth and development. Resources stored in seeds include carbohydrates and lipids to meet the energy demands of tissue construction and maintenance respiration, as well as nitrogen (N) and other minerals necessary for various metabolic activities (Mayer & Poljakoff Mayber 1982) The transition of dependency from seed reserve to external sources takes place at di fferent times for different resources, because the concentrations of various mineral nutrients in seeds are un (Fenner 1986; Kitajima 1996, 2002). Exclusive dependence on seed reserves ends earlier f or energy than for N (Kitajima 2002). E xperiments with single nutrient elimination from the growth medium have found N becomes exhausted from seed reserves first, both in legume and non leguminous temperate herbs (Fenner & Lee 1989; Hanley & Fenner 1997 ). Among essential mineral elements, N is particularly important and required in large quantities for construction of enzymes and nucleic acids, but also for certain defense chemicals such as alkaloids and non protein amino acids (Kursar et al. 2009). Typica l tissue N concentrations per unit dry mass range from 2% to 5%, but can to be as high as 8% in seeds (Barclay & Earle 1974) and from 1% to 3% in leaves (Roggy et al. 1999). N is stored in seeds as storage proteins, and it is exported to support seedling t issue construction (Mayer & Poljakoff Mayber 1982). Leaf construction requires high allocation of N, a large proportion of which (15 30%) exists in the key carboxylation enzyme, Rubisco (Evans 1989). Concentration and allocation of N vary substantially


58 amo ng organs within species, in relation to soil N availability, as well as light environment, all of which affect growth rate and N demands (Evans 1989). Between species differences in leaf N concentrations reflect not just N availability in the soil, but th e ecological strategy of the species (Poorter, Remkes & Lambers 1990). Recent community wide fertilizer addition experiments found that N and potassium (K) co limit growth of young trees in lowland moist tropical forest (Wright et al. 2011). In particular, l egumes are known for their high N demands which is considered to have evolved as a strategy for increasing photosynthetic rates, and resulted in an evolutionary pre condition that favors symbiotic relationships with N fixing bacteria ( McKey 1994 ). The h igh N demands of legumes may also favor high N allocat ion to seeds to meet the high N demands of young seedlings until they can start to acquire N from the environment with N symbiosis and pay for the energy demands from the symbiotic bacteria. Additionall y, N in the litter layer of forest floors may be limiting even in tropical forests with high N availability. In particular, shallow rooted plants, such as seedlings, in these forests are likely to be N limited (Hedin et al. 2009). It is unknown how long th e N demand of a developing seedling can be supported by seed reserves alone for tropical legumes or how allocation patterns may change in response to N demand for young seedlings. Here, I report the results of a shade house experiment in which I examined h ow seedlings of Balizia elegans (Fabaceae) respond to lack of N supply from external sources by comparing growth and biomass allocation of seedlings from contrasting soil N availability. Based on the results of Kitajima (2002) with three Bignoniaceae speci es, I predicted that seedlings of Balizia elegans would be able to grow for 2 3 weeks


59 following germination, after cotyledon s senesce, without external supply of N. After this period, however, I expected that seedlings without access to externally availabl e N should be smaller, containing lower tissue N concentrations, and allocate proportionally more biomass to roots compared to seedlings receiving external N. In addition, tissue N concentrations will also be greater in seedlings receiving external N. Meth ods Seeds from several individuals of Balizia elegans (Ducke) Barneby & J.W. Grimes (Fabaceae, Tribe Ingeae) were collected at La Selva biological station (Costa Rica, 10 ) in 2008. This species is a canopy emergent with relatively small seeds (0.042 g 0.008 g). In 2009, a subset of B. elegans seeds were clea ned (rinsed with 10% bleach solution followed by water), weighed, scarified to stimulate germination and planted in washed sand that was kept moist. Once seedlings matured their first leaf (fully expanded and green, approximately a month old), they were ra ndomly assigned to a nutrient treatment or harvested as part of a pre treatment group (used for initial biomass and nitrogen allocation measurements). Seedlings were grown in three replicated 2 m x 2 m shadehouses at ~10% photosynthetic photon flux density of full sun, the equivalent of a moderate sized gap (Chazdon & Fetcher 1984). This light level was chosen because juveniles of B elegans are typically found in areas of moderate light (Deborah Clark pers. comm.) and I expected seedlings would not be ligh t limited at this light level. The growth medium was a sand vermiculite mix (very low salt extractable N) and each seedling received 25 mL of either complete nutrient solution (all nutrient) or complete solution without N (no N) three times a week. The com


60 strength of the original formulation, which provided all nutrients required for plant growth including micronutrients (Table 4 1). In the no N solution, N was substituted from th e complete nutrient solution by replacing nitrate salts with chloride salts (KCl substituting KNO 3 ) and replacing the ammonium phosphate with potassium phosphate. Nutrient treatments began when seedlings had at least one fully expanded green leaf and cotyl edons senesced, rather than at a standard age after germination. The age of seedlings at the onset of the N treatments was 27 41 days (mean 38 3 days) after radicle emergence. Seedlings were harvested at three time intervals following the beginning of th e treatment: day 0, day 44, and day 57. At each harvest, I took standard measurements such as stem length and diameter, and leaf area. Harvested seedlings were separated into three organs (leaves, stems, and roots) ea were used to calculate leaf mass ratio (leaf mass/plant mass, LMR), root:shoot ratio (root mass/ shoot mass) and other measures of biomass allocation. Seedlings were pooled by shade house, harvest date and organ type before grinding them for determinati on of N concentration with an elemental analyzer (Costech Analytical ECS 4010, Valencia, CA). Nitrogen data were not available for the 57 day harvest. Multiplying tissue N concentration with the individual organ mass, I calculated the total N pool in each organ per plant, and summed these to calculate the total N pool in the seedling. Most of the data were not normally distributed, and hence I used Kruskal Wallis to test for treatment effects on response variables. All data were analyzed with JMP 8 and/or 9 (SAS institute).


61 Results By 44 days there was a clear difference in the leaf area and total biomass between the seedlings that received N and those that did not (Tables 4 2, 4 3; Figs. 4 1, 4 2). Leaf area increased little from the 0 day to 44 day harves t in no N treatment seedlings (the mean changed from 6.2 to 7.8 cm 2 ), even though the total seedling mass showed a significant increase (the mean changed from 27.4 to 59.2 mg). In contrast, the all nutrient treatment seedlings showed strong growth both in leaf area and biomass (Figs. 4 1, 4 2). The seedlings that received N had root : shoot ratios similar to the pre treatment harvest, which were lower than seedlings that received no external N supply (Table 4 2). Compared to all nutrient seedlings, seedling s in the no N treatment had higher leaf mass per area (LMA), root mass ratio (RMR, root mass/ whole plant mass) and LMR. Leaf, stem and root N pool increased in all nutrient treatment seedlings receiving N, but not in seedlings in the no N treatment (Table 4 2). In pre and all nutrient treatments, leaves had higher N concentrations than stems and roots, but the seedlings in the no N treatment had higher N concentrations in roots than in leaves and stems (Fig 4 3). Within each N treatment group there was no difference between the 44 and 57 day seedlings for any variable measured. Discussion Seedlings were ~ 5 weeks old at the onset of the nutrient treatments, by which point they probably had initiated autotrophic resource acquisition. This was not surprising given they had dropped their cotyledons and developed fully green leaves. Under the pre and no N treatments that supplied no external N, seedlings had similar leaf areas, indicating that seedlings had exhausted all N available in seeds to construct leaf area. Leaf area growth rates show feed forward responds to soil nutrient limitation,


62 slowing down before biomass growth ( Ingestad & Argen 1991; Ericsson 1995; Kitajima 2002). In a similar study of young legume seedling growth, leaf area growth declined sig nificantly in response to lack of N supply about 3 days before biomass growth responded (Martijn Slot and Danielle Palow, unpublished data). Nutrient limitation can affect plant biomass allocation patterns (Chapin 1980). A recent meta analysis of the effe cts of nutrient limitation on plant biomass allocation found a large increase in the allocation of biomass to roots when nutrients were limiting (Poorter et al. 2012). Here N limitation resulted in changes in biomass allocation, with proportionally more bi omass allocated to below ground parts rather than leaves, as commonly observed as higher root : shoot ratio and lower LMR (e.g., Fenner & Lee 1989; Ericsson 1995; Walters & Reich 2000). But, not all studies found significant effects on biomass allocation i n long term fertilization studies in the tropics (Burslem Grubb & Turner 1995; Gunatilleke et al. 1997). Additionally, RMR was greater under the no N treatment, similar to the findings of Fenner & Lee (1989) in a single nutrien t deprivation study and Juli ana, Burslem & Swaine (2009) in a nutrient addition study. Optimum tissue N concentrations should vary in relation to availability of N, as well as other environmental factors. As found by a meta analysis of tropical tree seedling response to nutrient sup ply, seedlings receiving N had higher N concentrations in shoots (Lawrence 2003). Furthermore, N concentrations found in the current study were lower than found in another study of initial seedling resource allocation which included B. elegans (Palow unpub lished, Chapter 3): nearly 50 mg/g less for all organ types. This may indicate that even at the beginning of this experiment seedlings had already diluted tissue N concentrations to the typical values for vegetative organs. Another study using


63 a tropical l egume found N concentration decreased ~50 mg/g in leaves and ~30 mg/g in stems, 30 days after reaching the first green leaf stage (Slot and Palow unpublished). It is unlikely that the tissue N concentrations achieved under the all nutrient treatment were t he result of luxury consumption, because they were lower than N concentrations o f the pre treatment seedlings. Surprisingly, leaf and root N concentration were not significantly different as found by others (Burslem et al 1995). Although N is generally c onsidered not to be limit ing of plant growth in lowland tropical forests ( Hedin et al 2009 ), recent evidence indicates that N may at least co limit growth of small trees (Wright et al 2011) and legumes are known for their high demand for N ( McKey 1994 ). It is clear from this study and others ( Fenner & Lee 1989 ; Kitajima 2002 ) that seed N is not sufficient to support seedling growth for much longer than several weeks under moderate to high light availability. It is possible that legume seedlings may need o nly enough N to survive until they form an association with their symbionts, and this strategy may have resulted in seedlings in the no N treatment allocating more N to their roots in preparation for nodule formation. Yet, Fenner & Lee (1989) found that le gumes generally respond to N limitation the same as other species. Young seedlings of Balizia elegans clearly need to acquire N from the environment at a relatively young age (< 3 months). Due to slow germination I was unable to conduct weekly harvests b ecause of the small sample size available. Weekly harvests would have allowed me to get a better picture of the timing of N limitation and changes in growth and allocation. However, I was able to see that N is important for the growth of B. elegans seedlin gs and they are able to survive solely on th e N from their seeds for more than 3 months following germination


64 Table 4 solution. This solution also contains micronutrients. Nutrie nt All treatment No N treatment NO 3 1.1 mM 0 mM NH 4 0.15 mM 0 mM K 0.6 mM 0.6 mM Ca 0.4 mM 0.4 mM P 0.3 mM 0.3 mM Mg 0.1 mM 0.1 mM S 0.1 mM 0.1 mM Table 4 2. Trait means (standard error) for all treatments and harvests. Days in treatment 0 44 44 57 57 Harvest 0 1 1 2 2 Treatment Pre treatment All nutrients No nitrogen All nutrients No nitrogen Sample size 9 12 12 12 13 Leaf area (cm 2 ) 6.2 (1.1) 38.5 (3.8) 7.8 (0.9) 41.0 (4.5) 9.11 (1.2) Leaf mass (mg) 14.9 (2.4) 100.7 (11.0) 29 .8 (4.5) 105.9 (11.7) 34.4 (4.4) Stem mass (mg) 6.1 (1.0) 29.7 (3.2) 15.4 (2.4) 33.8 (4.8) 19.0 (2.9) Root mass (mg) 6.4 (1.3) 29.9 (4.0) 14.0 (2.4) 29.2 (4.0) 20.7 (3.6) Total mass (mg) 27.4 (4.5) 160.3 (17.5) 59.2 (9.1) 169.0 (19.7) 7 4.1 (10.3) LMA (mg/mm 2 ) 0.025 (0.002) 0.026 (0.001) 0.037 (0.002) 0.026 (0.003) 0.037 (0.002) RS (g/g) 0.28 (0.03) 0.23 (0.03) 0.29 (0.02) 0.23 (0.02) 0.38 (0.03) LMR (g/g) 0.56 (0.03) 0.62 (0.02) 0.51 (0.02) 0.61 (0.03) 0.47 (0.03) Leaf N pool (mg) 0.55 (0.09) 3.19 (0.35) 0.51 (0.07) Stem N pool (mg) 0.20 (0.03) 0.61 (0.08) 0.24 (0.04) Root N pool (mg) 0.22 (0.05) 0.87 (0.12) 0.31 (0.05)


65 Table 4 3. Results of Kruskal Wallis test among nitrogen treatment groups. Because there was n o difference between 44 and 57 day harvests (time in treatment), the data from both harvests were pooled within each nitrogen treatment group. Letters represent the different treatment gr oups: a = all nutrients, n = no N. Variable Df p value Direction of significant effect Total leaf area 2 < 0.0001 (a > n) Total seedling biomass 2 < 0.0001 (a > n) Root:shoot ratio 2 0.0002 (a < n) Leaf mass ratio 2 < 0.0001 (a > n) Root mass ratio 2 0.0002 (a < n) Leaf N pool 2 < 0.0001 (a > n) Stem N pool 2 0 .0002 (a > n) Root N pool 2 0.0002 (a > n) Seedling N pool 2 < 0.0001 (a > n) LMA 2 < 0.0001 (a < n) Leaf N concentration 2 0.0273 (a > n) Stem N concentration 2 0.0390 (a > n) Root N concentration 2 0.0273 (a > n)


6 6 Figure 4 1. Total leaf ar ea (cm 2 ) of B. elegans seedlings (mean se) at different number of days after nutrient solution treatments. Symbols: diamond: all nutrient treatment (a), square: no N treatment (n), closed circle: pre pretreatment (p). Figure 4 2. Total seedling bioma ss (g) of B. elegans seedlings (mean se). See figure 4 1 for more details.


67 Figure 4 3. Nitrogen concentration (%) in organs for seedlings harvested at the beginning (p) and after 44 days under all nutrient (a) and no N (n) treatments. Means s.e.


68 C H APTER 5 CONCLUSIONS In this dissertation I use a functional trait approach to compare species in the tribe Ingeae, a tropical legume clade. This approach allow s me to explore how suites of traits evolved in this rapidly evolving group, with a special emph asis on how suites of traits may evolve with niche specialization to contrasting soil fertilities. Specifically, I examine the role of soil type association of functional traits at both the sapling and adult stages. Additionally, because the regeneration s tage is particularly important for population and community dynamics, I attempt to identify general patterns in regeneration traits using seed size (mass) as a key trait with which nitrogen (N) and phosophorus concentrations which may evolved in associati on (Chapter 2), and test effects of external nitrogen (N) on resource allocation traits of young Balizia elegans seedlings (Chapter 3) Soil type association influence s traits at both sapling and adult stages; species that prefer less fertile soils ha ve sui tes of traits that indicate the syndrome of slow growth and resource conservation through low tissue turnover rates. The patterns in the leaf functional traits and wood density are consistent with the expected differences of habitat filtering in resource r ich vs. less rich environments. The biomass and nutrient allocation patterns in adult and sapling leaves are generally associated with slow growth as a life history strategy. These differences in traits among species are at least partially explained by spe cies soil type preferences. Soil type preference also influence s seed nitrogen concentration and biomass in the nine species for which soil preference could be examined quantitatively. Interestingly, species specializing in the less fertile soil ha ve seeds with high N


69 concentrations, indicating possible natural selection for increased maternal investment of nutrient s to the offspring in environments in which seedlings face a greater challenge in soil nutrient acquisition. However, when a larger number of sp ecies are examined few clear patterns of resource allocation in relation to seed resources emerge. The only significant correlations are the positive correlation between seed and seedling stem nutrient concentrations, and the negative correlation of seed m ass and the proportion of seed biomass transferred to the seedling. Seedling leaves ha ve exceptionally high N concentrations compared to adult leaves, indicating temporary N enrichment from preferential transfer of seed N reserves to seedling leaves. Overa ll, the lack of correlation of seedling traits with seed traits may reflect differences in how selective pressures act on nutrient allocation patterns during initial seedling development. Seedlings of B. elegans allocate biomass and nitrogen differently in response to external nitrogen supply. When nitrogen i s not present, seedlings allocate proportionally more resources to roots than above ground organs. These plastic responses in allocation patterns follow the universally generalizable response to a limit ing nutrient (Poorter et al. 2012). Additionally, it i s apparent that B. elegans seedlings ha s sufficient seed resources to survive without external N for at least three months. Despite their high demand for N, legumes in the tribe Ingeae respond to enviro nmental conditions in a similar manner to those of other species. Various biotic and abiotic factors may be responsible for evolution of functional traits in Ingeae. My study indicates that soil type specialization explains at least part of the functional trait variations within a speciose genus Inga as most of the leaf, stem and seed traits examined show differences between specialists to alluvial vs. less fertile


70 residual soils. Overall, the results based on a species rich tribe of tropical legumes agr ee with the widely accepted perspective that considers both habitat filtering and phylogeny to play a role in trait evolution.


71 APPENDIX A ADDITIONAL TABLES AND FIGURE FROM CHAPTER 2 Table A 1. Means of adult traits by soil type for the three species tha t were collected on both soil types at La Selva Biological Station. See Table 2 1 for soil type preferences. Numbers in () indicate the number of individuals collected on residual/alluvial soil type for leaf traits (n = 10 across the soil type) and wood sp ecific gravity (n = 5 across the soil types) Species Trait Residual Alluvial t ratio p I. leiocalycina (5/5) LMA (g/m 2 ) 92.3 84.4 0.76 0.47 Leaf thickness (mm) 0.19 0.18 0.72 0.49 Leaf density (g/cm 3 ) 0.49 0.46 0.58 0.58 Foliar % N 3.31 3.55 2.44 0.04 Foliar % P 0.15 0.14 0.18 0.86 (2/3) Wood SG (g/cm 3 ) 0.56 0.47 3.49 0.07 I. alba LMA (g/m 2 ) 82.1 74.8 1.08 0.31 (5/5) Leaf thickness (mm) 0.19 0.18 0.96 0.37 Leaf density (g/cm 3 ) 0.43 0.41 0.43 0.68 Foliar % N 3.71 3.76 0.30 0.77 Foliar % P 0.16 0.17 1.10 0.30 (4/1) Wood SG (g/cm 3 ) 0.52 0.51 I. oerstediana LMA (g/m 2 ) 89.3 81.9 0.77 0.46 (4/6) Leaf thickness (mm) 0.32 0.29 0.91 0.39 Leaf density (g/cm 3 ) 0.28 0.28 0.14 0.90 Foliar % N 3.50 3.60 0.47 0.65 Foliar % P 0.17 0.17 0.04 0.97 (1/4) Wood SG (g/cm 3 ) 0.50 0.47


72 Table A 2. Means for leaf and sapling traits of nine Inga species at La Selva Biological Station, Costa Rica, including LMA (leaf mass per area), l amina T (lamina thickness), l amina D (lamina dr y mass density), h eight (mean adult height), DBH (diameter at breast height), and Narea (leaf N per unit lamina area). See Table 1 for species names. Letters that follow mean values indicate the results of post hoc Tukey Kramer pairwise comparisons. R = re sidual, A = alluvial Adults Saplings Species rank Soil pref. LMA (g/m 2 ) Lamina T (mm) Lamina D (g/cm 3 ) Height (m) DBH (cm) N area (g/m 2 ) LMA (g/m 2 ) Lamina T (mm) Lamina D (g/cm 3 ) N area (g/m 2 ) 1 R 100.8 a 0.35 a 0.29 de 24.9 24.8 2.38 bc 55.0 a 0.20 a 0.28 bc 1.45 bc 2 R 55.9 d 0.15 c 0.37 bc 14.7 12.3 1.73 d 3 R 98.9 a 0.30 a 0.34 cde 21.6 21.7 2.71 ab 57.5 a 0.19 ab 0.31 ab 1.56 abc 4 R 78.4 bc 0.19 b 0.42 ab 35.3 62.9 2.63 ab 55.3 a 0.17 bc 0.33 a 1.66 ab 5 R 74.2 bc 0.18 b 0.40 abc 26.3 33. 2 2.29 bc 50.1 ab 0.15 c 0.34 a 1.58 ab 6 none 88.3 ab 0.19 b 0.47 a 27.6 36.5 2.84 a 46.5 b 0.15 c 0.32 ab 1.58 abc 7 A 84.9 ab 0.30 a 0.28 e 23.3 33.0 2.63 ab 51.2 ab 0.18 ab 0.29 abc 1.50 bc 8 A 63.4 cd 0.22 b 0.29 e 19.8 21.6 2.41 abc 46.2 b 0.17 ab 0.27 c 1.78 a 9 A 65.4 cd 0.19 b 0.35 bcd 22.1 32.3 2.16 cd 45.3 b 0.18 ab 0.26 c 1.33 c mean R 83.3 0.24 0.36 2.39 54.5 0.17 0.31 1.56 mean A 71.2 0.24 0.31 2.40 47.6 0.17 0.27 1.54


73 Figure A 1. Phylogenetic relationships among 19 speci es in Ingae (including nine Inga species in the study plus those in other genera that occur in La Selva). For Inga species, genus name is not shown, and for species for which sufficient number of individuals were encountered in the survey, the species name is followed by soil habitat preference (R: residual, A: alluvial, N: no preference) and % of individuals encountered in the residual soil in the survey (Table 1). The tree was constructed from multiple plastid markers and the ITS nuclear marker by Dr. Kyl e Dexter using publicly available sequences (see Genbank Accession numbers in Kursar et al. 2009 and Dexter et al. 2010, plus accessions HM800435, GQ428675, FJ038512, and AY944538 for Abarema and AY944538, EF638186, and EU811973 for Cojoba ). Each tree repr esents the post burn in maximum clade credibility tree from a phylogenetic analysis using BEAST v1.54 (Drummond et al. 2007), with 10 million generations. Branch lengths are in terms of number of substitutions. These figures show qualitatively that best ph ylogenetic information available does not suggest any phylogenetic clustering of residual species to be likely among the study species. They also show that the two Zygia species are likely to be the closest relatives to Inga spp within Ingeae


74 APPENDIX B A DDITIONAL TABLE AND FIGURES FROM CHAPTER 3 Table B 1. The r esults from an ANOVA test comparing seed and seedling traits between species and species nested within study site Df=degrees of freedom. Trait Species Site (Species) df Seed mass <0.001 0.55 22 Seed N concentration <0.001 0.22 22 Seedling leaf N <0.001 <0.001 12 Seedling stem N <0.001 0.0123 12 Seedling root N <0.001 0.62 12 Seedling cotyledon N <0.001 0.42 12 Seed P concentration <0.001 17 Seedling leaf P <0.001 7 Seedling stem P <0.001 7 Seedling root P <0.001 7 Seedling cotyledon P 0.0031 7


75 Figure B 1. Mean seed nutrient concentrations (mg/g) plotted against seed mass for each mother tree. Triangles are data from BCNM and circles are data from LS, and closed symbols are Inga species. A. Seed N concentration (mg/g). B. Seed P concentration (mg/g).


76 Figure B 2 M ean seedling organ N (A) and P (B) concentration (mg/g dry mass) for each mother tree plotted against N concentration in seed excluding seed coat The three organ type s are leaves (closed), stem (open), and roots (grey). The lines are regression lines for the organ types: leaves (black), stem (dashed) and roots (grey).


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87 BIOGRAPHICAL SKETCH Danielle Therese Palow grew up in Miami, Fl orida surrounded by subtropical flora During her childhood she attended nature and scie nce camps that fostered her interest in biology. After g raduating from the Mast Academy, Science Magnet HS in 1996, s he began studying b iology at the U niversity of Miami. While at University of Miami she took a few courses in tropical biology, including a course in tropical ecology that culminated in a week long hike along an altitudinal transect in Costa Rica with David Janos. This course convinced her to pursue a graduate degree in tropical biology. Following graduation with a BS in 1999, Danielle took so me time off from education and volunteered at Fairchild Tropical Garden for the endangered species horticulture program. During her time at Fairchild she became interested in seedling biology. In tropical seedl ing ecology at Florida International University with Steve Oberbauer. After recei ving a worked for two months as a fiel d assistant for Chad Husby on a project in Chile and Argentina. In the summer of 2005, she began her research at Univ ersity of Florida with Kaoru Kitajima. During her tenure at University of Florida she has pursued her interest in tropical ecology, attending the Organization for Tropical Studies course in tropical ecology in 2006 and conducting research in Panama in 2006 and Costa Rica 2007 2010 In February 2012 she was resource faculty for the Organization for Tropical Studies fundamentals of tropical ecology course. She received her Ph.D. from the University of Florida in the spring of 2012.