The role of soil communities in the habitad partitioning between two morphospecies of the neotropical pioneer tree trema micrantha in central Panama

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(1)The role of soil communities in the habitat partitioning between two morphospecies of the neotropical pioneer tree Trema micrantha in Central Panama. Camila Pizano. UNIVERSIDAD DE LOS ANDES FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGIA BOGOTA D.C 2003.

(2) ABSTRACT Partitioning of species across resource gradients is though to play an integral role in the maintenance of species diversity in tropical forests. Recent studies (from the Barro Colorado Nature Monument in Panama) provide clear evidence of the existence of two morphospecies of what was known as the pioneer tropical tree Trema micrantha. These show a clear habitat partitioning between them; a small seeded morphospecies is found growing mainly on landslides along the shoreline of Lake Gatún, while a large seeded morphospecies shows preferences for treefall gaps in the forest. Moreover, ongoing genetic work strongly suggests that these morphospecies represent separate species. Neither light conditions nor dispersal limitation explain the differences in distribution between the two plant populations. However, soil conditions may be responsible for the different distributions of the two Trema morphospecies. Therefore, the purpose of this study was to determine the role of soil environments and communities in the adaptation of morphospecies to landslides and gaps. Both Trema morphospecies grew significantly better with the inoculation of arbuscular mycorrhizal fungi (AMF) in the greenhouse, and were found to be colonized by these fungi in the field. Furthermore, Trema morphospecies responded differently to the same soil type and inoculum source, each one growing better on its respective soil and soil community. Landslide Trema was more sensitive to different soil communities, showing a higher dependency on AMF and a lower resistance to pathogens. Gap Trema, showing a lower dependency on AMF and a higher resistance to pathogens, responded more strongly to abiotic properties of soil, specifically nutrient content. AMF spore densities were found to be similar between habitats, spore communities had higher species richness in gaps compared to landslides. This study shows that niche differentiation between Trema morphospecies may be determined and maintained by their adaptation to specific soil environments and soil microbial populations.. RESUMEN En estudios recientes (en el Monumento Natural de Barro Colorado) se ha reportado que existen dos morfoespecies del árbol tropical pionero Trema micrantha. Estas presentan, además, claras preferencias de hábitat. Una primer morfoespecie con semillas pequeñas ocurre principalmente en barrancos de las costas del lago Gatún, mientras que una segunda morfoespecie con semillas. 1.

(3) más grandes se encuentra exclusivamente en claros del bosque. Según los análisis genéticos realizados en el genero Trema, estas dos poblaciones representan diferentes especies (Garwood, et al., sin publicar). Ni las condiciones de luz, ni la limitante de dispersión explican las diferencias en distribución de las dos morfoespecies. Como consecuencia, el propósito de este estudio era determinar el papel de las propiedades abióticas y de las comunidades microbianas del suelo en la adaptación de las dos morfoespecies de Trema a barrancos y claros. Para empezar, se encontró que ambas morfoespecies de Trema son colonizadas por micorrizas arbusculares en el campo, y que las dos crecen mejor cuando son inoculadas con estos hongos en el invernadero. Mas aún, las morfoespecies de Trema respondieron de manera diferente al mismo inoculo y tipo de suelo, cada una creciendo mejor que la otra bajo sus propias condiciones ambientales. La morfoespecie que ocurre en los barrancos es más sensible a cambios en la comunidad del suelo, y mostró una mayor dependencia a las micorrizas y baja resistencia a los patógenos de raíces. Mientras tanto, la morfoespecies de los claros es menos dependiente a las micorrizas, más resistente a los hongos patógenos del suelo, y más responsiva a diferentes tipos de suelo (específicamente al contenido de nutrientes en el suelo). Adicionalmente, las comunidades de esporas de micorrizas resultaron ser más diversas en los claros que en los barrancos, a pesar de que ambos hábitats tienen similares contenidos de esporas. De esta manera, este estudio demuestra que la diferenciación de nichos entre las morfoespecies de Trema puede estar determinada y mantenida por su adaptación a diferentes comunidades microbianas y tipos de suelo.. 2.

(4) INTRODUCTION Habitat partitioning (e.g. moisture, light, nutrient) enhances the coexistence of species and maintenance of diversity in tropical plant communities (Grubb, 1976; Grubb, 1977; Denslow, 1980; Orians, 1982; Brokaw, 1985; Brokaw, 1987). Factors like competition between plants, and the interaction of plant species with the environment (climate, soil, herbivores, pathogens, seed dispersers and pollinators) are important determinants of plant community composition that contribute to the maintenance of life-history diversity. For ecologically similar species, divergence in one or a few traits may form the basis of niche differentiation (Dalling et al., 2003). Such is the case for pioneer species, well known for their crucial role in regeneration of forest disturbances. Early work on these plants established that they are functionally equivalent because of their similar growth requirements (Denslow, 1987). However, later and more detailed work shows that interspecific variation in dispersal, as well as seedling establishment, success and growth characteristics determine and promote the maintenance of diversity in this functional group of plants (Dalling and Hubbell, 2002). Trema micrantha (L.) Blume, a tropical pioneer tree, provides a model system for understanding niche differentiation in tropical forests. Recent work from central Panama showed that there are two distinct populations of this tropical tree and that there is a clear habitat partitioning between them. A small-seeded morphospecies occurs mainly on landslides along the shoreline of the Panama Canal, while a larger seeded morphospecies (endocarp almost three times larger) is almost exclusively found in large treefall gaps in old-growth and secondary forest (Silvera et al., 2003). Analyses based on molecular data and endocarp and leaf pubescence morphology indicate that these are indeed different species (Yesson et al., unpublished), and suggest Trema as a model taxon for understanding the evolution and speciation of tropical pioneer trees. A comparative study by Silvera et al (2003) on Barro Colorado Island showed that the relative growth rates (RGR) of Trema morphospecies do not differ across different light conditions, and that although their germination requirements are different (Table 1), the mechanism for habitat partitioning remains poorly understood. Light and water have been traditionally considered for understanding niche differentiation between different plant species. However, factors other than light, such as soil. 3.

(5) conditions, may play a key role in the habitat partitioning between the two Trema morphospecies found in central Panama (Antonovics and Bradshaw, 1970; Antonovics et al., 1971; Bradshaw, 1984). Adaptation to specific abiotic soil conditions has already been reported for other Trema species: Trema orientalis (L.) Blume is adapted to poor soils and dramite overburdens in India (Samantaray et al., 1995) and Trema tormentosa (Roxb.) Hara shows sensitivity to nutrient supply (Turner, 1991). In addition, other studies have reported the adaptation of plant species to specific ion and nutrient concentrations (Antonovics et al., 1971; Wu and Antonovics, 1975; McGraw et al., 1989), different soil microbial communities (Streitwolf-Engel et al., 1997), or both soil types and soil communities (Schultz et al., 2001). Although the heterogeneity in abiotic soil properties may predict plant species distributions, the interaction between plant species and soil communities has also been found to be crucial in determining plant community composition (Grime et al., 1987, Klironomos, 2002). Different microbial species are present in rhizosphere soil (defined as the volume of soil adjacent to and influenced by plant roots; Metting, 1993), where they establish dynamic interactions with plants. Pathogenic fungi, bacteria, and nematodes can reduce growth, reproduction and survival of plants (Burdon, 1987; Bruehl, 1987; Augspurger, 1990). Mean while, beneficial bacteria and mycorrhizal fungi benefit plants mainly by facilitating and increasing the uptake of minerals (primarily N by bacteria, and P by mycorrhizal fungi) (Albrecht et al., 1981; Smith and Read, 1997) and suppressing plant pathogens (Thomashow and Weller, 1990; Handman et al., 1991; Newshman et al., 1995a). Arbuscular mycorrhizae fungi (AMF; class Zygomycetes, order Glomales) establish symbiotic associations with the roots of about 85% of all terrestrial plant species (Malloch et al., 1980; Janos, 1980a). These fungi benefit plants by improving the uptake of poorly mobile soil nutrients (mainly phosphorus (Smith and Read, 1997), but also nitrogen (Tobar et al., 1994), potassium and copper (Habte and Manjunath, 1991)), limiting the uptake of toxic heavy metals (Gildon and Tinker, 1983), providing protection against pathogens (Newsham et al., 1995; Borowicz, 2001), and drought resistance (Safir et al., 1972; Sieverding, 1991; Levy and Kriken, 1980; Allen et al., 1981; Hardie and Leyton, 1981; Allen, 1982). In addition, AM fungi may alter the foraging strategies of plants (affecting clonal growth) (Streitwolf-Engel et al., 1997), as well as the outcome of interspecific plant competition (Fitter, 1977; Allen, 1991; Watkinson and Freckleton, 1997). The host plant in return, gives carbon compounds to the fungi.. 4.

(6) The complex dynamics between plant and soil communities involve the interactions within plant and soil microbial communites, as well as how much both entities affect each other. Particular plant species directly influence the composition of the rhizosphere microbial population (Westover et al., 1997). Free-living bacterial communities (Westover, 1995) and the relative growth rates of fungal populations (Bever et al., 1996) are host dependent. For example, plant identity significantly influences spore abundances and species composition of arbuscular mycorrhizal (AM) fungal communities (Johnson et al., 1997; Bever et al., 1996; Eom et al., 2000). In the same way, soil microorganisms have the potential to affect plant performance, influencing plant community composition (Grime et al., 1987). Plant competitive ability has shown to be highly dependent on the particular mycorrhizal associations some plants establish (Fitter, 1977; Hartnett et al., 1993; Watkinson and Freckleton, 1997). In addition, plant performance has proved to vary across different arbuscular mycorrhizae fungal communities (Gange, 1993; Kiers et al., 2000; Bever, 2002a). A feedback model that predicts the outcome of the interaction between plants and soil microorganisms has been proposed and extensively discussed and tested by Bever and colleagues (Bever, 1994; Bever et al., 1997; Bever,1999; Bever et al., 2000; Bever et al., 2002; Bever, 2002a; Bever, 2002b). According to this model (Box 1), relative growth rates of plants and microbial populations are mutually interdependent (Bever, 1999). In this way, plant and soil microorganisms’ performance depends upon the specific plant-soil community combination. The feedback process may be viewed in the following way. A plant or plant population changes the soil community, and this change in turn affects the rate of growth of that plant or plant population (Bever, 1997). Two outcomes arise from the possible directions and strength of this interaction: positive and negative feedback (Bever, 1994). Positive feedback arises when a particular soil community promotes the growth of a given plant, which in return benefits that microbial population. As a consequence, the presence of that plant species would benefit its associated soil community and vice versa. If for any reason that plant species becomes more abundant, it would indirectly (by benefiting its soil community) increase its own growth rate relative to that of other plant species. As a result, local diversity of the plant community would decrease. However, positive feedback may help establish spatial structure of the plant and soil microorganism populations, contributing to the maintenance of plant diversity at larger scales (Bever et al., 1997; Molosky et al., 1999).. 5.

(7) On the other hand, negative feedback occurs when there is an asymmetric interaction between plants and their soil communities. In this case, a particular plant or plant population does not promote the growth of its own soil community, but, indirectly, the one growing on other plants’ roots (e.g. Kiers et al., 2001). Consequently, if the frequency of the first plant increases, it would indirectly promote the growth rate of a second plant species. As a result, negative feedback can contribute to the maintenance of plant diversity at a local scale. The plant-soil community feedback model may be applied to different plant and soil microorganism associations (Bever, 1994). For example, there is a positive feedback between legumes and nitrogen-fixing bacteria (Nutman and Read, 1952; Robinson, 1969; Lie et al., 1987; Chanway et al., 1988) as well as in clover and Rhizobium (Mytton, 1975), and a negative feedback has been demonstrated in agricultural systems (Shipton, 1977; Cook, 1981; Kollmorgen et al., 1985; Gerhardson, 1992) and temperate perennial plants (Bever, 1994). Indeed, the maintenance of diversity through community feedback has been mainly tested for plant-arbuscular mycorrhizal fungal mutualism (Bever, 1999; Eom et al., 2000; Bever et al., 2001; Kiers et al., 2001; Bever et al., 2002,; Bever, 2002a; Bever, 2002b). In addition to plant community composition (Gange, 1993; Van der Heijden et al., 1998a), plant diversity, variability, and productivity (Van der Heijden et al., 1998b; Klironomos et al., 2000) have been shown to be influenced by arbuscular mychorrhizae fungi distribution and diversity. Although often considered ecologically equivalent and functionally redundant, AM fungal species not only differ in how beneficial (and costly) they are for particular plants, but also in their response to different plant species and soil habitats (reviewed by Bever et al., 2001). Co-occuring endomycorrhiza fungal species are functionally diverse and selective for host plants (Sanders, 1993; Streitwolf-Engel et al., 1997; Helgason et al., 2002). Therefore, if certain plant species respond differently to specific AM fungal species, then, the diversity and spatial distribution of different AM fungal species can generate environmental heterogeneity that promotes the maintenance of plant diversity (Newham et al., 1995b; Bever et al., 2001, Kiers et al., 2001). Indeed, several studies in temperate systems have shown that plant diversity (Newman and Redell, 1988; Gange et al., 1993; Van der Heijden et al., 1998b) and productivity (Klironomos et al., 2000) are enhanced by a highly diverse community of endomycorrhizae fungi.. 6.

(8) Plant diversity is particularly high in lowland tropical forests, where soils generally have low fertility and limited P availability favoring the establishment of mycorrhizal associations (Janos, 1983; Newshman et al., 1995b). A high proportion of tropical plants are, in fact, mycotrophic (Janos, 1980a; Janos, 1980b; Carneiro et al., 1996), and their related endomycorrhizal communities have been found to be highly diverse and variable in scale and time (Janos,1992; Husband et al., 2002a; Husband et al., 2002b). In addition, plant dependency on AMF infection is related and varies across successional plant groups (Siqueira et al., 2001). Pioneer species, with small seeds, cannot depend upon their reserves during early stages of initial growth, and therefore, are the most responsive to AMF root colonization (compared to early secondary, late secondary and climax species) during early life stages (Siqueira et al., 1998). Zangaro et al. (2000) have shown that tropical pioneer trees have the highest dependency on AMF, highest root colonization by AMF in the field, and highest inoculum potential (number of mycorrhizal associations formed as defined by Tommerup, 1992) among all four successional groups of plant (Zangaro et al., 2000). Trema micrantha is a pioneer tree that has been found to be highly dependent on AMF (Carneiro et al., 1996; Siqueira et al., 1998; Zangaro et al., 2000; Siqueira et al., 2001). As a consequence, Trema morphospecies may be adapted to particular soil conditions (e.g. Antonovics and Bradshaw, 1970; Antonovics et al., 1971; Bradshaw 1984) facilitated by their association with AMF communities. For example, a recent study by Schultz et al. (2001) shows that two ecotypes of the temperate grass Andropogon gerardii has adaptated to different soil nutrient levels by a shift in their dependence on AMF and root architecture. Plants adapted to poor soils have coarse roots and high dependency to mycorrhizal fungi, while plants occurring on richer soils have branched roots and lower dependency on AMF. In a similar way, different soil communities and specifically, AMF species may mediate the adaptation of Trema morphospecies to the different soil conditions of landslides and gaps. Landslides and treefall gaps have often been compared because they are both disturbances that promote early succession. However, some plant species (mostly pioneers) almost exclusively benefit from landslide openings (Whitmore, 1975; Herwitz, 1981; Guariguata, 1990), due to their differential nutrient availability, water capacitance, soil stability, and arbuscular mycorrhizal contents (Dalling, 1994). In addition, landslides, unlike gaps, have impoverished seed banks, poor dispersal (of both seeds and fungal propagules), differential. 7.

(9) activities of seed predators, and altered soil moisture and temperature regimes (Dalling and Tanner, 1995). Consequently, soil environments and communities are different between gaps and landslides (Janos, 1980a). Spore number and root density differ from one habitat to the other (Janos,1992). The proportion of spore content may be higher in landslides, where there are less roots in general, and proportionally more dead radicular tissue liberating spores. Inoculum from treefall gaps, on the other hand, should be mainly composed of “runner” hyphae from live roots (Janos, 1990), some hyphae from dead roots, and a constant input of spores dispersed by rodents (Mangan and Adler, 1999; Mangan and Adler, 2002). Interestingly, spore number (Picone, 2000) as well as inoculum potential, are higher in areas at the beginning of succession than in forests (Fischer et al., 1994; Zangaro et al., 2000). Trema morphospecies are discretely (with no intermediate forms bewteen them) distributed in landslides and treefall gaps on Barro Colorado Nature Monument (in Panama) (Silvera et al., 2003). In a greenhouse experiment simulating different gap sizes, Silvera et al (2003) found similar relative growth rates for the two morphospecies across different light gradients, suggesting that light does not explain the mechanism of habitat partitioning between morphospecies. However, Trema growing on landslides has a higher specific leaf area (SLA) and unit leaf rate (ULR), indicating that this is a more light demanding morphospecies. In addition, morphospecies have significantly different endocarp masses; Trema occurring in gaps has an endocarp that is nearly three times larger than that of the landslide morphospecies. In spite of this, since fruits are similar in size, small seeded morhospecies tends to have a proportionately higher investment of total fresh fruit mass in mesocarp tissue compared to the large-seeded morphospecies. A summary of the differences between the Trema morphospecies is given in Table 1. Dispersal limitation may explain the absence of large-seeded morphospecies on landslides, but it does not explain the lack of small-seeded morphospecies in treefall gaps (Silvera et al., 2003). Landslides have an impoverished seed bank; therefore, since large-seeded morphospecies persists in the soil for years and germinates from the seed bank, it is unlikely that it ever colonize such habitats. However, a smaller endocarp mass is generally associated with a higher fecundity and consequently, a dispersal advantage. Therefore, Trema growing on landslides should be able to colonize gaps as well as landslides.. 8.

(10) In disturbances like forest gaps and landslides, what determine seed germination and seedling establishment are the competitive interactions between different plant species, and the interaction of plants with their soil environment (Walker et al., 1996). Given that uptake of P plays a key role for plant regeneration in both habitats (Fetcher et al., 1996; Frizano et al., 2002), plants that have adapted to landslides or gaps should be adapted to their AMF communities as well. Trema morphospecies face exactly this situation, and habitat parititioning between them may be mediated by their adaptation to different soil environments through the association with particular AMF (and other microbial) populations. To elucidate the mechanism of niche differentiation between the two morphospecies of Trema, in this study I first examined if both morphospecies associate with different AMF communities in the field. Then I tested the effect of specific soil conditions on the perfomance of both morphospecies in the greenhouse. My specific objectives were to: 1). quantify AMF root colonization for Trema morphospecies in the field, 2). compare AMF spore communities between landslides and gaps, 3). test the response of both morphospecies to AMF colonization, and 4). determine the effect of landslide and gap soil and soil communities on Trema morphospecies growth.. METHODS. Study site and species This study was conducted in a seasonally moist lowland tropical forest, on Barro Colorado Nature Monument (BCNM) in Central Panama (9°10´N, 70°51´W). The reserve consists of Barro Colorado Island (BCI) and its adjacent islands and peninsulas in Gatun lake. Rainfall averages 2600 mmy¯¹, with a severe dry season from December to April or early May (Leigh, 1999). Croat (1978), and Foster and Brokaw (1982) have described the flora and vegetation of the area, while geology and hydrology have been characterized by Dietrich et al. (1982). Trema is a pantropical genus of fast-growing short-lived pioneer trees. Trema micrantha (L.) Blume is the most widely distributed and variable taxon, occurring from Florida to northern Argentina, across different habitats. It is found in montane as well as dry forests, and from sea level up to 2800m in elevation (Garwood et al., unpublished). It is a high-light demanding. 9.

(11) pioneer tree (2-20m high) that occurs in largely disturbed areas, and produces flowers and fruits throughout the year. It is monoecious, with very small, petal-less, greenish-yellow flowers and 34mm globose drupes that turn red when ripe. At BCNM, Trema seeds are primarily dispersed by birds (Silvera et al., 2003). Two distinct populations of Trema micrantha with significantly different seed germination behavior and habitat preference have been found and described in Central Panama (Garwood et al., unpublished). The first population occurs mainly in treefall gaps in the interior of the forest (henceforth called “gap Trema”), has deep physiological seed dormancy (persists in the soil for many years), and temperature-dependent germination. The second population is almost exclusively found on landslides along the shore of the Panama Canal (henceforth called “landslide Trema”), and has a quick light- dependent germination. In addition, Trema occurring in gaps have black, larger, prominently ridged endocarp with irregular pores, while Trema growing on landslides have smaller, brown endocarp, with cratered surface and smooth pores. Analyses based on allozymes, chloroplast sequence and morphological characters (endocarps and leaf pubescence) indicate that the two Trema populations found in Central Panama are separate species (Yesson et al., unpublished).. Quantification of AMF root colonization in Trema and spore community description in the field Sixty-two Trema individuals from different ages were found and marked across Barro Colorado Island. From these, 34 were large-seeded Trema growing in 14 gaps of different ages, and the other 28 belonged to the small-seeded morphospecies growing on 6 different landslides. From each individual, 200g of fine roots and rhizophere soil (10-30cm) where collected from four different locations around the tree’s root system. Fine roots were washed with running water and short segments were cleared in 10% KOH and 5% H202 acidified with HCL 1%, washed with running water and stained with 0.05% tryplan blue in lactoglycerol solution (Kormanik and McGraw, 1982; Zangaro, et al., 2000). Fungal colonization was measured and quantified in the following way. Selected root segments were mounted on slides (2 slides/individual and 15 root segments/slide), and presence or absence of AMF colonization was recorded at 200 intersect points for each slide (for a total of 400 intersect points for each individual) (Nicolson, 1960; Daft and Nicolson, 1966; Crush, 1973; Giovannetti and Mose, 1980) to record colonization percentage (as hyphae, arbuscules or vesicules) for each Trema individual. AM fungal spores. 10.

(12) were extracted from 10g dry weight soil subsamples using wet-sieving and sucrose density gradient centrifugation (Daniels and Skipper, 1982). The extracted spores were observed, counted and identified under a light microscope by Dr. Ahn-Heum Eom. Only the viable spores (based on color, shape, surface conditions and examination of spore contents) were included. Total spore number, spore species and diversity were recorded for each of the sites.. Greenhouse experiment To determine the effect of soil communities on Trema morphospecies performance, a pot experiment with three factors was set up in the greenhouse. The three factors were: type of soil (landslide or gap), inoculum source (sterile, landslide and gap), and Trema morphospecies (landslide Trema and gap Trema). There were twelve treatments combining the three mentioned factors, and 4 additional ones used to test the effect of inoculation with AMF (sterile vs. live inoculum), and nutrient addition (no nutrient addition vs.complete nutrient supplement) on Trema morphospecies’ performance. In that way, there was a total of 16 treatments, 8 replications each, assigned to random locations in the greenhouse. Eight of those treatments were used to test the response of the two Trema morphospecies to nutrient addition and inoculation with AMF (common soil type×two Trema morphospecies×nutrient or not nutrient addition×sterile or inoculated with AMF). The other eight treatments tested the response of morphospecies to different combinations of soil type and inoculum source (landslide or gap soil×two Trema morphospecies×landslide or gap inocula). For soil types, soil was collected from five different landslides and five different gaps, mixed with washed sea sand (60% soil, 40% sand) and sterilized in individual bags in the autoclave for one hourbag¯¹. For the inoculum, rhizosphere soil and fine roots were collected from different Trema trees in five forest gaps and five landslides. For “forest” inoculum, equal portions of homogenized root segments and rhizosphere soil from the five gaps were mixed, and the same was done for the “landslide” inoculum. The 1gal pots where inoculated with 250ml of such inoculum (for the sterile treatments the inoculum was autoclaved but still used). All pots received the same amount of light (30.6% from the light reaching the greenhouse) and water. For the fertilized treatments, Bayfolan Bayer (N, P2O5, K2O, S, B, Co, Zn, W, Mo, Ca, Mn, Fe and Mg) was used at 1.0 ml/l. 11.

(13) concentration and 50 ml of the solution were applied to each plant under nutrient treatment every week. Pots were randomly re-distributed every ten days. Plants were grown for 90 days and leaf area of each plant was measured every two weeks. Leaf area and wet and dry weights were recorded after harvesting. Root systems were stained as described above and scoled for AMF presence (or absence, in the case of sterile treatments).. Data Analysis Total AMF colonization was compared between Trema individuals from gaps (n=34) and landslides (n=28). All data were normalized by Arcsin, and a t-test (SYSTAT 10) was made to compare AMF colonization percentages (as hyphae, arbuscules and vesicules individually, and as total colonization) between the two Trema populations. A log transformation was used to compare the number of other structures present in the roots of the two Trema morphospecies. Finally, total number of spores and total number of AMF species were compared between landslides (n=15) and gaps (n=16). All means were compared by using a t-test (SYSTAT 10). Data from the greenhouse experiment was transformed in the following way: total dry weight and Relative Growth Rate (RGR) of plants was log transformed, and an Arcsin transformation was applied to the Root/Shoot Ratio. The sixteen treatments were separated to address different questions. First, the effects of nutrients and inoculation with live soil on Trema morphospecies were analyzed across the eight treatments combining the two morphospecies, the two inoculum states (sterile vs. inoculation with live soil), and the nutrient contents in the soil (no nutrient addition vs nutrient addition). Second, the effects of habitat soil “conditions” on Trema morphospecies were analyzed across the treatments that included the two morphospecies growing on either landslide or gap soil conditions (both inoculum and soil from a particular habitat). Finally, the data from the twelve treatments combining the two morphospecies, the two soil types, and the two inoculum sources was analyzed to examine the response of Trema morphospecies to different soil types and inoculum sources combinations. In this last analysis the sterile treatment was not included, but is included in the figures for comparison. For all of the above, an ANOVA was made using the General Linear Model Procedures (GLM) in SPSS.. 12.

(14) RESULTS Quantification of AMF root colonization and spore community description in the field Individuals of different ages of both Trema morphospecies from landslides and gaps were colonized by arbuscular mycorrhizae fungi in the field. Landslide Trema roots showed marginally higher percentages of AMF colonization, but hyphae (P=0.574), arbuscules (P=0.202), vesicules (P=0.051), and total colonization (P=0.173) percentages did not differ significantly between the two Trema morphospecies in their habitats (Fig 1). In addition to AMF, there were other unidentifiable structures present in the roots of Trema morphospecies in both gaps and landslides. The number of these structures was significantly higher in the roots of gap Trema than in the roots of the morphospecies occurring on landslides (Table 2), indicating that gaps have a richer soil community. AMF spore number did not differ between habitats (P=0.812), though there were higher numbers of AMF species associated with gap Trema than with the landslides morphospecies (Table 2). Also, there were differential abundances of particular AMF species between habitats, and some fungal spores were found exclusively in only one of the two soil communities (Table 3). For example, various Acaulospora and Glomus species were more common in landslides than in gaps, while the spores of Scutellospora and Glomus species were more abundant in gaps.. Greenhouse experiment Effect of nutrients and AMF inoculation on Trema morphospecies growth Gap Trema had higher relative growth rate (RGR) (P<0.05) and total biomass (P<0.001) than landslide Trema across different treatments. For both morphospecies, the addition of nutrients and AMF inoculum increased plant growth (Table 4, Figs. 3a and 3b), having higher biomass (Fig. 2a, P<0.001) and RGR (Fig. 2b, P<0.001) when inoculated with AMF (live or unsterilized inoculum) regardless of soil nutrient content. With and without nutrients, all plants grew bigger and faster with AMF, having the highest RGR when both AMF and nutrients were added to the soil (P<0.001). Nutrient addition by itself (under sterile conditions), however, only. 13.

(15) increased the biomass of the gap morphospecies (P<0.01) and had no significant effect on landslide morphospecies. Nutrient addition and inoculation with AMF also changed the biomass allocation patterns of the two Trema morphospecies (Fig 3c). In general, both morphospecies produced more roots in sterile soil relative to inoculated soil (P<0.001). This shift in biomass allocation was particularly clear in landslide Trema, which had a significantly lower root:shoot ratio when it was inoculated with AMF (P<0.001) regardless of the soil nutrient contents. In contrast, the effect of AMF inoculation on biomass allocation in gap Trema shifted with nutrient addition. Without nutrients, this morphospecies responded in the same way as landslide Trema to AMF inoculation. However, with the addition of nutrients, this morphospecies produced proportionately more roots when inoculated with AMF than under sterile conditions (P<0.001). Trema morphospecies response to landslide and gap soil conditions (soil type and inoculum source) Trema morphospecies responded differently to the same soil type and inoculum source (P<0.05), each one growing better on their respective soil conditions (Table 5, Fig. 3). Accordingly, landslide Trema had a significantly higher biomass (Fig. 3a) and RGR (Fig. 3b) compared to gap morphospecies under landslide conditions. In contrast, gap Trema grew significantly bigger and faster than landslide morphospecies with gap soil and inoculum. Gap Trema allocated significantly more biomass to roots than landslide morphospecies in both landslide and gap soil conditions (Fig. 3c, P<0.01). Trema morphospecies response to different soil types and inoculum sources Both soil type and inoculum source had an effect on Trema morphospecies performance (Table 6, Fig. 4). In general, both morphospecies had significantly higher total biomass (Fig. 4a) and RGR (Fig. 4b) with landslide inoculum (total biomass, P<0.001; RGR, P<0.01) regardless of soil type, and with forest soil (total biomass, P<0.001; RGR, P<0.001) across different inoculum sources. All plants had the highest total biomass with landslide inoculum and gap soil, and the lowest total growth with landslide soil and gap inoculum (P<0.05). Following a similar pattern, biomass allocation to roots (Fig. 4c) was significantly higher for all plants with gap inoculum regardless of soil type (P<0.05), and with landslide soil. 14.

(16) regardless of inoculum source (P<0.001). Furthermore, both morphospecies produced the highest amount of roots when they grew on landslide soil and gap inoculum (P<0.001). DISCUSSION Quantification of AMF root colonization and spore community description in the field Arbuscular mycorrhizal fungi occur at similar levels in the roots of Trema individuals from both landslides and treefall gaps. Percentages of root colonized by hyphae, arbuscules and vesicules did not differ significantly between morphospecies (Fig.2). Mycorrhizal colonization was marginally higher in the roots of landslide Trema, but this pattern may be due to the higher content of tannins in the roots of gap morphospecies (personal obs.) that limited the view of all fungal structures in the root cells. As a consequence, AMF colonization percentages may be underestimated for the roots of Trema from the gaps. These results suggest that both Trema morphospecies invest similar carbon quantities in maintaining the fungi in their roots (Graham et al., 1991; Graham et al., 1997), although they do not necessarily predict how AMF affect plant performance (Janos, 1987). Both Trema morphospecies associate with arbuscular mycorrhizal fungi in gaps and landslides, where the acquisition of limiting P is particularly important (Frizano et al., 2001). My results show that there is a similar number of AMF spores associated with Trema individuals in the two habitats, although their commmunity composition differed between habitats. AMF spore populations were found to be spatially heterogeneous (Janos, 1992), and the fungi seemed to establish non-random associations with the two different hosts (Husband et al., 2002). Gap AMF spore communities had a higher species richness compared to landslides. Also, the abundance of particular AMF species differed between soil communities, and some species exclusively occurred in just one of the two habitats (Table 3). For example, Acaulospora genus and some Glomus species were more abundant in spore populations of landslides, while Scutellospora genus and other Glomus species were more common in gaps. Differences between AMF spore communities may be due to several factors. On one hand, there is a limited dispersal of propagules (including spores) to landslides compared to forest gaps (Dalling, 1994), and some mycorrhizal species that are mainly dispersed by rodents (Janos, 1980b; Mangan and Adler, 1999; Mangan and Adler, 2002) are unlikely to arrive at landslides (e.g. Glomus fasciculatum). Root density is also lower in landslides compared to gaps,. 15.

(17) thus limiting the dispersal of AMF from plant to plant by hyphae (Janos, 1992; Janos, 1980a; Janos, 1980b). Second, arbuscular mycorrizhal fungi species are adapted to specific mineral environments in the soil, and vary in life history characters such as dormancy, germination and sporulation requirements, and resistance to extreme soil conditions (Bever et al., 2001). Hence, differences in abiotic factors between gap and landslide microsites may be responsible for the differential distribution of AMF species among habitats. For example, some AMF species may not survive in landslides where the soil moisture is low (Dalling, 1994), the substrate is unstable (Nakanuro, 1984; Guariguata, 1990), the temperatures are high (Dalling, 1994), and there is a low nutrient status (Flaccus, 1959; Adams and Sidle, 1987; Guariguata, 1990; Fetcher et al., 1994). In addition to AMF, there are other microorganisms (pathogenic fungi and bacteria, beneficial bacteria, and other fungi) that occur in soil communities and also colonize roots, affecting plant species. The number of other unidentified soil microorganisms was significantly higher in the roots of gap morphospecies compared to the Trema that grows on landslides (Table 2). This suggests that gap Trema maintains a higher number of both AMF and other soil microorganism species in its roots. As discussed above, this may be due to a higher dispersal of propagules and more favorable abiotic conditions in gaps than in landslides. Thus, these results show for the first time, that AMF are equally abundant in both landslides and gaps, that AMF communities are different between these two habitats, and that soil communites are richer in gaps than in landslides. These differential soil microhabitats may be favor the establishment of particular plant species, as it may be the case for Trema morphospecies.. Greenhouse experiment Effect of nutrients and AMF inoculation on Trema morphospecies growth Both nutrient addition and AMF inoculation increased plant growth and RGR of landslide and gap Trema morphospecies (Table 4, Fig. 2). However, the perfomance of gap Trema was mainly increased by nutrient addition, while landslide Trema responded more to inoculation with AMF. In addition, biomass allocation also suggests that this morphospecies is more dependent on endomycorrhizal fungi. Gap morphospecies had a higher root/shoot ratio when colonized by mycorrhizal fungi (compared to the sterile treatments), indicating that it is less dependent on. 16.

(18) mycorrhizal fungi (Azcón and Ocampo, 1987). Gap Trema may be highly efficient for obtaining soil nutrients by having a particular root architecture (Newham et al., 1995). In contrast, landslide Trema allocated more biomass to roots under sterile conditions probably due to a restricted root system (Manjunath and Habte, 1990; Newham et al., 1995) that is particularly efficient when colonized by AMF. Consequently, this morphospecies may have adapted to landslides, where nutrients are limited (Allen, 1991; Gange et al., 1993; Fetcher et al., 1996; Frizano, 2002), by associating and depending on arbuscular mycorrhizal fungi. However, to fully evaluate AMF dependency of a plant species, both AMF and host species have to interact across a gradient of established soil solution P concentrations (Habte and Manjunath, 1991), and that hasn’t been done. Trema morphospecies response to landslide and gap soil conditions (soil type and inoculum source) Trema morphospecies responded differently to landslide and gap soil type and inoculum, each morphospecies having both a higher total biomass and RGR when grown on its respective soil conditions (Table 5, Fig. 3). Most notably, only on landslide soil and inoculum landslide Trema had a higher RGR than gap Trema. These results indicate that morphospecies are sensitive to specific soil environments, benefiting from particular combinations of soil mineral availability and mycorrhizal fungi (Janos, 1980b; Van der Heijden et al., 1998a; Schultz et al., 2001). Moreover, they show that there is a positive feedback between each Trema morphospecies and their respective soil communities (Bever et al., 1997; Bever, 1999; Kiers et al., 2001; Bever et al., 2002,{ Bever, 2002a; Bever, 2002b), suggesting that habitat partitioning between the two Trema morphospecies is likely mediated by morphospecies’ adaptation to particular soil conditions. This is the first study showing that partitioning between light demanding species may be mediated by the soil niche. Trema morphospecies response to different soil types and inoculum sources Trema morphospecies responded differently to particular soil types and inoculum sources (Table 6, Fig. 4), and this may explain how they benefit from their respective habitat soil conditions. Landslide Trema was more responsive to inoculum sources, growing significantly better with landslide inoculum across different soil types. This morphospecies may have adapted to landslide limited mineral availability (Guariguata, 1990; Dalling, 1994) by associating and. 17.

(19) depending on mycorrhizal fungi (Janos, 1980b; Gange et al., 1993; Frizano et al., 2002). Therefore, it may be highly adapted to landslide soil community, growing better with the particular array of soil microorganisms that occur in the soil of that habitat (specifically AMF) (Van der Heijden et al., 1998a; Bever et al., 2001). In contrast, gap Trema responded more to different soil types, growing better with gap soil regardless of inoculum source. This morphospecies may have a lower dependency on mycorrhizal fungi compared to landslide Trema, since there is a higher nutrient availability in treefall gaps than in landslides (Denslow, 1985; Denslow et al., 1998). Therefore, gap Trema may be adapted to the particular abiotic properties of gap soils but not to a particular array of soil microorganisms (Janos, 1980a; Janos, 1980b; Schultz et al., 2001). Landslide inoculum and gap soil was the most favorable soil environment for both Trema morphospecies. In contrast, all plants allocated more biomass to roots, and grew the less with the combination of landslide soil and gap inoculum. Accordingly, in nature, landslides offer the most beneficial inoculum and the poorest soil, while gaps offer the richest soil and the less beneficial inoculum for Trema morphospecies. Soils are poor in landslides (Dalling, 1994; Fetcher et al., 1996), where some plants allocate a high proportion of biomass to roots for obtaining limiting nutrients (Clarkson, 1985; Manjunath and Habte, 1990). In addition, soil microbial communities are less rich in landslides than in gaps (see the first section of the results). Therefore, there may be a lower accumulation of pathogens in landslide soils, where the substrate is unstable and the dispersal is limited (Dalling and Tanner, 1995). Conversely, gaps have a high income of propagules of different fungal species (Mangan and Adler, 2002) and may accumulate a higher content of soil pathogens as a result of having a stable substrate. Consequently, and probably due to the antagonistic effects of these microbes, both Trema morphospecies grew shoddier with inoculum from the gaps (Anderson et al., 1994). However, gap inoculum had stronger negative effects on the growth of landslide Trema than on the plants of gap Trema, indicating that gap morphospecies may have a higher resistance to hostile soil microorganisms than landslide morphospecies. Finally, this may be related to the higher content of tannins in the roots of gap Trema, which provide resistance to soil pathogens.. 18.

(20) MYCORRHIZAL FUNGI-PATHOGEN DRIVEN HYPOTHESIS This study demonstrated that habitat partitioning between the two Trema morphospecies is likely mediated by their adaptation to different soil types and soil mircrobial communities. Landslides have limited nutrient availability, low dispersal of arbuscular mycorrhizal fungi and pathogens, unstable substrate, low content of roots, and high soil temperatures. In contrast, gaps have higher contents of nutrients in the soil, higher dispersal of propagules, and a stable substrate. Accordingly, soil abiotic conditions and soil microbial populations differ between the two habitats. Gap soils, rich in nutrients, contain highly diverse soil communities, in which both AMF and soil pathogens accumulate. Therefore, given that gap soil has high nutrient availability, gap Trema associates with mycorrhizal fungi but does not depend heavily on these fungi. This was reflected in the greenhouse experiment, where gap Trema was highly responsive to nutrient addition and particular soil types. Additionally, this morphospecies was not affected by different inocula, and showed no response to the antagonistic effects of gap soil microbes. On one hand, the roots of gap Trema have a high content of tannins that provide protection against pathogents. On the other hand, given that there is a high dispersal of fungal propagules to treefall gaps, there is a constant income of fungal species to these habitats, and soil microbial communities are highly variable from one gap to another. Therefore, gap Trema could not evolve dependency to a specific array of microbial species in soil communities. Landslide soils have low nutrient availability, poorly diverse mycorrhizal communities, and low accumulation of pathogens. Consequently, landslide Trema depends on AMF for obtaining limiting nutrients, as my results showed. This morphospecies grew significantly better, maximizing its growth above ground when inoculated with mycorrhizal fungi. In addition, landslide Trema was highly sensitive to different inoculum sources, and its growth was diminished by the hostile soil microbes from gaps. This pattern may be related to two factors. First, since there is a lower accumulation of pathogens in the soil of landslides, landslide Trema has not evolved resistance to their attack. Second, given that AMF communities are less diverse in landslide soils, these fungi conform similar arrays of species across different landslides. Consequently, landslide morphospecies, having a high dependency on mycorrhizal fungi, has also evolved dependency to particular arrays of arbuscular mycorrhizal species.. 19.

(21) AKNOWLEDGEMENTS I want to thank my advisors (Jim Dalling, Allen Herre and Santiago Madriñán), Scott Mangan and Dr. Ahn-Heum Eom for their unconditional and extremely valuable advice and intelligence; Arturo Morris, Evelyn Sanchez, Eloisa Lasso and Sebastian Bernal for assisting my greenhouse experiment; BCI crew for helping me setting up my experiment and bringing soil from the field; Catalina Londoño and Maria Caridad García for their generosity and crucial initial guidance in the study of AMF, and the reviewers of this thesis (Matthew Gilbert, Allen Hurlbert, Jonathan Myers, Kathleen Rudolph, Keryn Bromberg and James Mandel). This study was funded by Allen Herre, Jim Dalling, and the Short-Term Fellowships program in the Smithsonian Tropical Research Institute.. REFERENCES Adams, P.W. and R.C. Sidle. 1987. Soil conditions in three recent landslides in S.E. Alaska. Forest Ecology and Management, 18: 93-102. Albrecht, S.L., Y. Okon, J. Lonnquist and R.H. Burton. 1981. Nitrogen fixation by cornAzospirillium associations in a temperate climate. Crop Science, 21: 301-306. Allen, M.F., W.K. Smith, T.S. Moore, and M. Christensen. 1981.Comparative water relations and photosynthesis of mycorrhizal and non-mycorrhizal Bouteloua Gracilis (H.B.K.) Lag ex Stend. New Phytologist, 88: 686-693. Allen, M.F. 1982. Influence of vesicular–arbuscular mycorrhizae on water movement through Bouteloua Gracilis. New Phytologist, 91: 191-196. Allen, E. B and M.F. Allen. 1986. Water relations or xeric grasses in the field: interactions of mycorrhizas and competition. New Phytologist, 104: 559-571. Allen, M.F. 1991. The ecology of mycorrhizae. Cambridge: Cambridge University Press.pp. 105120. Allen, E.B., E. R., M. F. Allen, A. Pérez-Jimenez and P. Huante. 1998. Disturbance and seasonal dynamics of mycorrhizae in a tropical decidious forest in Mexico. Biotropica, 30(2): 261274.. 20.

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(32) TABLES AND FIGURES Table 1. General characteristics of the two Trema morphospecies found in BCNM (Garwood et al., unpublished; Yesson et al., unpublished; Silvera et al., 2003). Landslide morphospecies. Gap morphospecies. Distribution. Landslides along the lake shore. Gaps in the interior of the forest. Endocarp mass (mg). 1.38 (±0.13). 3.83 (±0.22). Endocarp color. Brown. Black. Endocarp surface. Cratered, with smooth pores. Prominently ridged, with irregular pores. Specific Leaf Area. Lower. Higher. Unit Leaf Rate (ULR). Higher. Lower. Light demand. Higher. Lower. Seed germination. Light-dependent. Temperature-dependent. Seed dormancy. None. Deep physiological seed dormancy. Presence in seed banks. Not common. Very common around BCNM. Response to AMF. Very high (probably highly. High. (SLA). dependent on AMF) Response to different. Very high. Medium. Low. Very high. soil communities Response to nutrient contents in the soil. 31.

(33) Table 2. F-values for analyses of variance conducted on the number of other microorganisms, AMF spores, and AMF species found to be associated with Trema morphospecies in gaps and landslides. All dependent variables were log10-transformed prior to analysis. * stands for a statistical significance of P<0.05 and *** stands for a statistical significance of P<0.001. Landslide Trema. (n=15) Gap Trema (n=16). F. 5. 9. 7.28***. Total number of AMF spores. 5023. 6006. 0.216. Average AMF species richness. 8.53. 11. 2.26*. Total number of other microorganisms present in the roots. Table 3. Species of arbuscular mycorrhizal fungi that were found exclusively in the spore community associated with Trema morphospecies individuals from gaps (n=16) and landslides (n=15). Gaps. Landslides. Glomus clavisporum. Acaulospora denticulata. Glomus sinuosum. Glomus tortuosum. Glomus sp. 3 (unkoown) Scutellospora calospora Scutellospora sp. bl (unknown) Scutellospora sp. wl (unknown) Scutellospora sp. y (unknown) Glomus rubiforme. 32.

(34) Table 4. F-values from analyses of variance conducted on the data from the greenhouse experiment with the two Trema morphospecies to test the effects of nutrient addition and AMF inoculation on plant performance (total growth (total dry weight), relative growth rate (RGR) and biomass allocation (root/shoot ratio). Total growth was log10-transformed and RGR and root/shoot ratio was Arcsin-transformed prior to analysis. * stands for a statistical significance of P<0.05, ** stands for a statistical significance of P<0.01, and *** stands for a statistical significance of P<0.001. Source. Total growth. RGR. Root/Shoot. Species. 17.061***. 4.307*. 41.162***. Nutrient addition. 2.979. 178.674***. 3.356*. Inoculation with AMF. 97.353***. 55.378***. 78.662***. Species*Nutrient addition. 9.296**. 1.319. 19.673***. Species*Inoculation with AMF. 0.053. 0.677. 20.192***. Nutrient addition*Inoculation with AMF. 0.436. 47.820***. 2.327. Species*Nutrient addition*Inoculation. 8.109**. 1.184. 22.789***. with AMF. Table 5. F-values from analyses of variance conducted on the data from the greenhouse experiment in which the effects of landslide and gap soil conditions (soil type and inoculum) were tested on plants of both Trema morphospecies performance (total dry weight, relative growth rate (RGR) and biomass allocation (root/shoot ratio)). Total growth was log10transformed and RGR and root/shoot ratio was Arcsin-transformed prior to analysis. * stands for a statistical significance of P<0.05 and ** stands for a statistical significance of P<0.01. Source. Total growth. RGR. Root/Shoot. Species. 1.161. 1.190. 8.491**. Condition. 4.096. 4.081. 3.344. Species*Condition. 5.835*. 5.843*. 0.014. 33.

(35) Table 6. F-values from the analyses of variance conducted on the data from the treatments of the greenhouse experiment in which the effects of different soil types (landslide and gap) and inoculum sources (landslide and gap) were tested on Trema morphospecies plant performance (total dry weight, relative growth rate (RGR) and biomass allocation (root/shoot ratio)). Total growth was log10-transformed and RGR and root/shoot ratio was Arcsin-transformed prior to analysis. * stands for a statistical significance of P<0.05, ** stands for a statistical significance of P<0.01, and *** stands for a statistical significance of P<0.001. Source. Total growth. RGR. Root/Shoot. Species. 2.465. 2.141. 1.393. Soil type. 59.873***. 34.675***. 19.710***. Inoculum source. 28.079***. 13.194**. 11.314*. Species*Soil type. 0.243. 2.434. 0.811. Species*Inoculum source. 4.669*. 1.299. 0.94. Soil type*Inoculum source. 9.683**. 0.003. 18.14***. Species*Soil type*Inoculum source. 0.764. 0.60. 0.286. 34.

(36) Box 1. Feedback model for the interaction of plant species and their respective soil communities. Possible outcomes resulting from the mutual interdependence of plants (A and B) and their respective soil communities (X and Y). The direction of benefit delivered between the two plant species and their soil communities are indicated in the arrows, their thickness indicating the magnitude of benefit. When the interaction between plants and their associated soil microbial populations is symmetrical (as presented in the figure on the left), a positive feedback results. In that way, if a particular plant species A becomes more abundant in the community, it will benefit soil community X, enhancing its own growth. Consequently, plant A will increase its abundance with time, probably excluding plant B from the community. On the other hand, when there is an asymmetrical interaction between plant and soil microbial populations, there is a negative feedback (as presented in the figure on the right). In this case, plant A favors soil community Y more than soil community X. Therefore, as this plant species becomes more abundant, it will enhance growth of plant B indirectly. As a result, negative feedback will be promoting the coexistence of plant species and the maintenance of diversity in the community (Bever, 1994).. 35.

(37) Gap sp. (n=34). 70. Landslide sp. (n=28). % Colonization. 60 50 40 30 20 10 0 Hyphae. Arbuscules. Vesicules. Total Col.. AMF structure Figure 1. Observed AMF root colonization percentages (in terms of hyphae, arbuscules, vesicules and total colonization) for Trema morphospecies in their natural habitats. Quantified percentages were not significantly different between landslide and gap morphospecies.. 36.