Peter G. Kennedy

Fungal Community Ecology: A Hybrid Beast with a Molecular Master

ABSTRACT Fungi play a major role in the function and dynamics of terrestrial ecosystems, directly influencing the structure of plant, animal, and bacterial communities through interactions that span the mutualism-parasitism continuum. Only with the advent of deoxyribonucleic acid (DNA)-based molecular techniques, however, have researchers been able to look closely at the ecological forces that structure fungal communities. The recent explosion of molecular studies has greatly advanced our understanding of fungal diversity, niche partitioning, competition, spatial variability, and functional traits. Because of fungis unique biology, fungal ecology is a hybrid beast that straddles the macroscopic and microscopic worlds. While the dual nature of this field presents many challenges, it also makes fungi excellent organisms for testing extant ecological theories, and it provides opportunities for new and unanticipated research. Many questions remain unanswered, but continuing advances in molecular techniques and field and lab experimentation indicate that fungal ecology has a bright future.

Potential link between plant and fungal distributions in a dipterocarp rainforest: Community and phylogenetic structure of tropical ectomycorrhizal fungi across a plant and soil ecotone

*Relatively little is known about diversity or structure of tropical ectomycorrhizal communities or their roles in tropical ecosystem dynamics. In this study, we present one of the largest molecular studies to date of an ectomycorrhizal community in lowland dipterocarp rainforest. *We sampled roots from two 0.4 ha sites located across an ecotone within a 52 ha forest dynamics plot. Our plots contained > 500 tree species and > 40 species of ectomycorrhizal host plants. Fungi were identified by sequencing ribosomal RNA genes. *The community was dominated by the Russulales (30 species), Boletales (17), Agaricales (18), Thelephorales (13) and Cantharellales (12). Total species richness appeared comparable to molecular studies of temperate forests. Community structure changed across the ecotone, although it was not possible to separate the role of environmental factors vs host plant preferences. Phylogenetic analyses were consistent with a model of community assembly where habitat associations are influenced by evolutionary conservatism of functional traits within ectomycorrhizal lineages. *Because changes in the ectomycorrhizal fungal community parallel those of the tree community at this site, this study demonstrates the potential link between the distribution of tropical tree diversity and the distribution of tropical ectomycorrhizal diversity in relation to local-scale edaphic variation.

Root tip competition among ectomycorrhizal fungi: Are priority effects a rule or an exception?

Competition for root colonization among ectomycorrhizal fungi is well documented, but the mechanisms determining competitive outcomes are not clearly understood. In a previous study, we observed that timing of colonization (i.e., a priority effect) had a significant effect on the outcome of competition between two ectomycorrhizal (EM) fungi in the genus Rhizopogon. In this study, we explicitly tested the role of priority effects in competition among EM fungi by experimentally manipulating the timing of colonization of four Rhizopogon species on Pinus muricata seedlings. In a first experiment, we set up 12 two-species combinations, in which seedlings were first inoculated from spores with one species, grown for three months, and then inoculated with an equal density of spores of a second species and grown for an additional three months. Root tip occupation in the two-species treatments was determined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of internal transcribed spacer region (ITS) of rDNA. In a second experiment, we further examined competitive interactions between two Rhizopogon species using split-root P. muricata seedlings. One side of the root system was pre-colonized by one species, and spores of the second species were added to the other side of the root system in all same and different species pair-wise combinations. We found that for three of the four species (R. occidentalis, R. salebrosus, R. vulgaris), the outcome of competition in the first experiment depended strongly on the timing of colonization, with the first colonizing species always being the competitive dominant. For R. evadens, however, initial colonization did not prevent significant subsequent colonization by R. occidentalis and R. vulgaris. This appeared to be caused by the lower colonization of R. evadens compared to the three other species. In the second experiment, we observed that the portion of the split root system that was initially uncolonized remained receptive to colonization when spores were added. The amount of colonization of R. occidentalis and R. salebrosus on the side of the root system to which they were added was not significantly influenced by species identity on the other side of the seedling. In combination, these results confirm that priority effects do play a major role in dynamics of EM root tip colonization, at least in the early colonization of seedlings, and that the proportion of the root system occupied by a species appears to be a key factor determining competitive success.

Ectomycorrhizal fungi and interspecific competition: species interactions, community structure, coexistence mechanisms, and future research directions

The field of ectomycorrhizal fungal (EMF) ecology has largely developed outside the ecological mainstream, owing in large part to the challenges in studying the structure and dynamics of EMF communities. With advances in molecular identification and other research techniques, however, there has been growing interest among mycologists and ecologists in understanding how different ecological factors affect EMF community structure and diversity. While factors such as soil chemistry and host specificity have long been considered important, an increasing number of laboratory and field studies have documented that interspecific competition also has a major impact on EMF species interactions and may significantly influence EMF community structure. In this review, I examine the progress that has been made in understanding the nature of EMF competition. Currently, there are four conclusions that can be drawn: negative competitive effects are rarely reciprocal; competitive outcomes are environmentally context-dependent; field distributions often reflect competitive interactions; and timing of colonization influences competitive success. In addition, I highlight recent studies documenting links between competitive coexistence and EMF community structure, including checkerboard distributions, lottery models, storage effects, and colonization-competition tradeoffs. Finally, I discuss several aspects of EMF competition needing further investigation and some newer methods with which to address them.

Biogeography of ectomycorrhizal fungi associated with alders (Alnus spp.) in relation to biotic and abiotic variables at the global scale

· Much of the macroecological information about microorganisms is confounded by the lack of standardized methodology, paucity of metadata and sampling effect of a particular substrate or interacting host taxa. · This study aims to disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhizal (ECM) fungi at the global scale by using comparable sampling and analysis methods. · Ribosomal DNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide. Use of spatial and phylogenetic eigenvectors along with environmental variables in model selection indicated that phylogenetic relations among host plants and geographical links explained 43 and 10%, respectively,in ECM fungal community composition, whereas soil calcium concentration positively influenced taxonomic richness. · Intrageneric phylogenetic relations among host plants and regional processes largely account for the global biogeographic distribution of Alnus-associated ECM fungi. The biogeography of ECM fungi is consistent with ancient host migration patterns from Eurasia to North America and from southern Europe to northern Europe after the last glacial maximum, indicating codispersal of hosts and their mycobionts.

SUPPLY‐SIDE ECOLOGY IN MANGROVES: DO PROPAGULE DISPERSAL AND SEEDLING ESTABLISHMENT EXPLAIN FOREST STRUCTURE?

Theory and empirical evidence suggest that spatial and temporal variation in propagule availability can have as great or greater an influence on community structure as post-recruitment biotic interactions, a phenomenon known as supply-side ecology. One of the first theories to invoke supply-side dynamics, D. Rabinowitzs Tidal Sorting Hypothesis (TSH), attributed the tidal zonation of mangrove tree species to the interacting effects of water depth and propagule size on dispersal and establishment. According to the TSH, smaller propagules are carried farther inland by flood tides than larger ones, stranding and establishing in greater numbers at upper tidal elevations. In contrast, larger propagules are better able to establish in deeper, more seaward areas because their size affords greater access to the soil surface and resistance to buffeting by moving water. Here we present results of the first comprehensive investigation of the TSH. In a series of experiments conducted at Punta Galeta on the Caribbean coast of Panama, we quantified patterns of propagule dispersal and establishment of the three dominant species: Avicennia germinans, Laguncularia racemosa, and Rhizophora mangle. To measure dispersal patterns, we monitored movements (directions and distances) of marked propagules released at three elevations along each of three transects. On all transects, regardless of species or elevation, propagules moved seaward rather than being carried inland by tides, as predicted by the TSH. Rates of seedling establishment were monitored in experiments conducted at different tidal elevations. Contrary to the TSH, all three species established best in the lower intertidal, where they were in prolonged contact with the soil surface during low tides, and established more poorly in the upper intertidal basin, where standing water made rooting difficult. Seedling establishment was lowest in the wave-exposed, back reef habitat, but in accordance with the TSH, the large propagules of Rhizophora rooted and persisted better in this turbulent habitat than did the smaller propagules of the other species. We conclude that the TSH does not explain the vertical distributions of mangrove species on Punta Galeta. Rather, a different form of supply-side dynamics imposed by the strong seaward movement of dispersing propagules is important in structuring our study forests.

Determining the outcome of field-based competition between two Rhizopogon species using real-time PCR

Interest in the ecology of ectomycorrhizal (ECM) fungi has increased considerably, but little is known about interspecific interactions among ECM species. We examined competitive interactions between Rhizopogon occidentalis and R. salebrosus at Point Reyes National Seashore, California, USA. At three field sites, species abundances were compared in single‐ and two‐species treatments on Pinus muricata seedlings inoculated with spores. Competition for root tips was assessed using real‐time polymerase chain reaction (PCR) of internal transcribed spacer rDNA. In general, we found strong competitive exclusion of R. salebrosus by R. occidentalis, with ≥ 75% of the seedlings in the two‐species treatment colonized exclusively by R. occidentalis after 5 and 10 months. However, on the seedlings that were co‐colonized, we observed no significant difference in the abundances of R. salebrosus and R. occidentalis, suggesting that once R. salebrosus was established, it was no longer competitively inferior. There were no significant differences in survival, growth, or percentage leaf nitrogen of seedlings colonized with either Rhizopogon species, but both growth and percentage leaf nitrogen were significantly higher for ECM than non‐ECM seedlings. We also observed strong positive correlations between actual ECM root tip weight and that inferred from real‐time PCR for both species, indicating that this method provided an accurate assessment of root tip occupation and hence ECM competitive dynamics. In conjunction with a previous experiment, our results indicate that competition between these two Rhizopogon species occurs similarly in both field and laboratory settings and that when colonizing from spore, timing largely determines the outcome of initial competitive interactions.

Ectomycorrhizal fungi in Mexican Alnus forests support the host co-migration hypothesis and continental-scale patterns in phylogeography

To examine the geographic patterns in Alnus-associated ectomycorrhizal (ECM) fungal assemblages and determine how they may relate to host plant biogeography, we studied ECM assemblages associated with two Alnus species (Alnus acuminata and Alnus jorullensis) in montane Mexico and compared them with Alnus-associated ECM assemblages located elsewhere in the Americas. ECM root samples were collected from four sites in Mexico (two per host species), identified with ITS and LSU rRNA gene sequences, and assessed using both taxon- (richness, diversity, evenness indices) and sequence divergence-based (UniFrac clustering and significance) analyses. Only 23 ECM taxa were encountered. Clavulina, an ECM lineage never before reported with Alnus, contained the dominant taxon overall. ECM assemblage structure varied between hosts, but UniFrac significance tests indicated that both associated with similar ECM lineage diversity. There was a strikingly high sequence similarity among a diverse array of the ECM taxa in Mexico and those in Alnus forests in Argentina, the United States, and Europe. The Mexican and United States assemblages had greater overlap than those present in Argentina, supporting the host–ECM fungi co-migration hypothesis from a common north temperate origin. Our results indicate that Alnus-associated ECM assemblages have clear patterns in richness and composition across a wide range of geographic locations. Additional data from boreal western North America as well as the eastern United States and Canada will be particularly informative in further understanding the co-biogeographic patterns of Alnus and ECM fungi in the Americas.

Different soil moisture conditions change the outcome of the ectomycorrhizal symbiosis between Rhizopogon species and Pinus muricata

The outcome of species interactions often depends on the environmental conditions under which they occur. In this study, we tested how different soil moisture conditions affected the outcome of the ectomycorrhizal symbiosis between three Rhizopogon species and Pinus muricata in a factorial growth chamber experiment. We found that when grown in 7% soil moisture conditions, ectomycorrhizal plants had similar biomass, photosynthesis, conductance, and total leaf nitrogen as non-mycorrhizal plants. However, when grown at 13% soil moisture, ectomycorrhizal plants had significantly greater shoot biomass, higher photosynthetic and conductance rates, and higher total leaf nitrogen than non-mycorrhizal plants. The differences in plant response by mycorrhizal status in the two soil moisture treatments corresponded with evidence of water limitation experienced by the fungi, which had much lower colonization at 7% compared to 13% soil moisture. Our results suggest that the outcome of the ectomycorrhizal symbiosis can be context-dependent and that fluctuating environmental conditions may strongly affect the way plants and fungi interact.

Propagule size and predispersal damage by insects affect establishment and early growth of mangrove seedlings

Variation in rates of seedling recruitment, growth, and survival can strongly influence the rate and course of forest regeneration following disturbance. Using a combination of field sampling and shadehouse experiments, we investigated the influence of propagule size and predispersal insect damage on the establishment and early growth of the three common mangrove species on the Caribbean coast of Panama: Avicennia germinans, Laguncularia racemosa, and Rhizophora mangle. In our field samples, all three species exhibited considerable intraspecific variation in mature propagule size, and suffered moderate to high levels of predispersal attack by larval insects. Rates of insect attack were largely independent of propagule size both within and among trees. Our experimental studies using undamaged mature propagules showed that, for all three species, seedlings established at high rates regardless of propagule size. However, propagule size did have a marked effect on early seedling growth: seedlings that developed from larger propagules grew more rapidly. Predispersal insect infestations that had destroyed or removed a substantial amount of tissue, particularly if that tissue was meristematic or conductive, reduced the establishment of propagules of all three species. The effect of sublethal tissue damage or loss on the subsequent growth of established seedlings varied among the three mangrove species. For Avicennia, the growth response was graded: for a propagule of a given size, the more tissue lost, the slower the growth of the seedling. For Laguncularia, the response to insect attack appeared to be all-or-none. If the boring insect penetrated the outer spongy seed coat and reached the developing embryo, it usually caused sufficient damage to prevent a seedling from developing. On the other hand, if the insect damaged but did not penetrate the seed coat, a completely healthy seedling developed and its growth rate was indistinguishable from a seedling developing from an undamaged propagule of the same size. Similar to Avicennia, if an infestation did not completely girdle a Rhizophora seedling, it survived, but grew at a reduced rate. In summary, our experiments demonstrated that natural levels of variation in propagule size and predispersal damage by insects translate into significant differences in seedling performance in terms of establishment and/or early growth. Such differences are sufficiently large that they could influence the intensity and outcome of competitive interactions during forest regeneration.