Environmental changes in biodiversity hotspot ecosystems of
South Ecuador: RESPonse and feedback effECTs (FOR2730)

Abstract:

Anthropogenic atmospheric deposition can increase nutrient supply in the most remote ecosystems, potentially affecting soil biodiversity. Arbuscular mycorrhizal fungal (AMF) communities rapidly respond to simulated soil eutrophication in tropical forests. Yet the limited spatio-temporal extent of such manipulations, together with the often unrealistically high fertilization rates employed, impedes generalization of such responses. We sequenced mixed root AMF communities within a seven year-long fully factorial nitrogen (N) and phosphorus (P) addition experiment, replicated at three tropical montane forests in southern Ecuador with differing environmental characteristics. We hypothesized (i) strong shifts in community composition and species richness after long-term fertilization, (ii) site- and (iii) clade-specific responses to N versus P additions depending on local soil fertility and clade life history traits respectively. Fertilization consistently shifted AMF community composition across sites, but only reduced richness of Glomeraceae. Compositional changes were mainly driven by increases in P supply while richness reductions were observed only after combined N and P additions. We conclude that moderate increases of N and P exert a mild but consistent effect on tropical AMF communities. To predict the consequences of these shifts, current results need to be supplemented with experiments that characterize local species-specific AMF functionality.

Abstract:

Global changes in nutrient deposition rates are likely to have profound effects on plant communities, particularly in the nutrient-limited systems of the tropics. We studied the effects of increased nutrient availability on the seedlings of six tree species in montane forests of southern Ecuador in situ. After five years of continued N, P, or N+P addition, naturally grown seedlings of each of the two most common species at each elevation (1000, 2000, and 3000 m asl) were harvested for analyses of leaf morphology, nutrient content, herbivory, and tissue biomass allocation. Most species showed increased foliar N and P concentrations after addition of each respective element. Leaf tissue N:P ratios of >20 in the control plants of all species suggest that P is more growth-limiting in these forests than N. Leaf morphological responses to nutrient addition were species and nutrient specific, with some species (Hedyosmum purparescens, Graffenrieda emarginata) exhibiting increased specific leaf area (SLA), and others (Graffenrieda harlingii) increased leaf area ratios (LAR). Pouteria torta (1000 m) had lower SLA and LAR after P addition. Increased herbivory was only evident in G. emarginata (after N and N+P addition). Only the species from 3000 m asl modified biomass allocation after nutrient addition. In general, N and N+P addition more strongly affected the species studied at the upper elevations, whereas P addition had a similar range of effects on the species at all elevations. We conclude that the responses of the studied tropical montane forest tree seedlings to chronic N and P addition are highly species-specific and that successful adaptation to increased nutrient availability will depend on species-specific morphological and physiological plasticity.

Abstract:

Recent studies raise the hypothesis that Na shortage restricts decomposition and affects the carbon cycle in tropical forests. When Na concentrations in soils are low and the stands are far off-coast, they do not receive substantial Na inputs from the atmosphere. Since terrestrial plants have low concentrations of Na, which is not considered as an essential element, the demand of soil fauna may not be covered. Yet, in contrast to animals, little is known of Na demands of fungi and phyllosphere microorganisms. We present results from a study on Na limitation in a montane forest ecosystem in South Ecuador, which is located on the eastern cordillera of the Andes. We tested the hypotheses that (1) the study area is characterized by low Na concentrations because of low deposition rates with incident precipitation (wind directions mainly from the Amazonian Basin), (2) decomposition processes are limited by fauna and fungal Na restrictions and (3) Na is retained in the canopy because of Na limitation of microorganisms in phyllosphere. Since 1998, we measure Na fluxes in rainfall, throughfall, stemflow, litter leachate, litterfall and organic layer in a microcatchment under an undisturbed lower montane rainforest. Results reveal comparatively low Na concentrations in the ecosystem and similar Na concentrations in throughfall and stemflow. Since Na fluxes are lower with throughfall than with incident rainfall, we conclude that Na is retained in the canopy. To explore the role of the phyllosphere in Na retention we sampled leaves covered by phyllosphere microorganisms and leaves without phyllosphere cover from several tree species, which were sprayed with a NaCl solution containing 0.5 mg L-1 Na, corresponding to the Na concentration in incident rainfall in our study area. Additionally, responses of litter decomposition to Na additions and the involved interaction of soil fungi and fauna were tested in a litterbag experiment at two sites (1000 and 2000 m a.s.l.). Results revealed enhanced decomposition rates following Na additions, though only in the presence of soil fauna. These results might have future ecosystem implications, since our time series showed that total Na deposition decreased within the past 15 years from ca. 40 kg ha-1 a-1 to 10 kg ha-1 a-1, suggesting a potential role of Na in regulating ecosystem processes.

Abstract:

Summary
Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities.
We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing.
The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community.
The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.

Abstract:

Tropical ecosystems have an important role in global change scenarios, in part because they serve as a large terrestrial carbon pool. Carbon protection is mediated by soil aggregation processes, whereby biotic and abiotic factors influence the formation and stability of aggregates. Nutrient additions may affect soil structure indirectly by simultaneous shifts in biotic factors, mainly roots, and fungal hyphae, but also via impacts on abiotic soil properties. Here, we tested the hypothesis that soil aggregation will be affected by nutrient additions primarily via changes in arbuscular mycorrhizal fungal (AMF) hyphae and root length in a pristine tropical forest system. Therefore, the percentage of water-stable macroaggregates (> 250 ?m) (WSA) and the soil mean weight diameter (MWD) was analyzed, as well as nutrient contents, pH, root length, and AMF abundance. Phosphorus additions significantly increased the amount of WSA, which was consistent across two different sampling times. Despite a positive effect of phosphorus additions on extra-radical AMF biomass, no relationship between WSA and extra-radical AMF nor roots was revealed by regression analyses, contrary to the proposed hypothesis. These findings emphasize the importance of analyzing soil structure in understudied tropical systems, since it might be affected by increasing nutrient deposition expected in the future.

Abstract:

Studies in temperate systems provide evidence that the abundance of arbuscular mycorrhizal fungal (AMF) depends on soil nutrient availability, which is mainly explained in the context of resource stoichiometry and differential plant biomass allocation. We applied this concept to an understudied ecosystem – tropical montane forest – analyzing root and AMF abundance along an elevational gradient with decreasing nutrient availability, combined with responses to nitrogen (N) versus phosphorus (P) additions. At three sites from 1000 to 3000 m above sea-level we analyzed fine root length, AMF root colonization as well as extraradical AMF biomass (neutral lipid fatty acid 16:1?5, hyphal length and spore counts) in a nutrient manipulation experiment. We found a significant increase in root length as well as intra- and extraradical AMF abundance with elevation. Overall, P additions significantly increased, whereas N additions decreased AMF abundance, with differential though nonsystematic changes along the elevational gradient. Strongest effects were clearly observed at the intermediate site. These findings suggest a general dependency of roots and AMF on nutrient availability, though responses to N and P additions differed from previous studies in temperate systems. In the context of future nutrient depositions, results suggest diverging responses of AMF abundance depending on site characteristics.

Abstract:

Increased nitrogen (N) depositions expected in the future endanger the diversity and stability of ecosystems primarily limited by N, but also often co-limited by other nutrients like phosphorus (P). In this context a nutrient manipulation experiment (NUMEX) was set up in a tropical montane rainforest in southern Ecuador, an area identified as biodiversity hotspot. We examined impacts of elevated N and P availability on arbuscular mycorrhizal fungi (AMF), a group of obligate biotrophic plant symbionts with an important role in soil nutrient cycles. We tested the hypothesis that increased nutrient availability will reduce AMF abundance, reduce species richness and shift the AMF community toward lineages previously shown to be favored by fertilized conditions. NUMEX was designed as a full factorial randomized block design. Soil cores were taken after 2 years of nutrient additions in plots located at 2000 m above sea level. Roots were extracted and intraradical AMF abundance determined microscopically; the AMF community was analyzed by 454-pyrosequencing targeting the large subunit rDNA. We identified 74 operational taxonomic units (OTUs) with a large proportion of Diversisporales. N additions provoked a significant decrease in intraradical abundance, whereas AMF richness was reduced significantly by N and P additions, with the strongest effect in the combined treatment (39% fewer OTUs), mainly influencing rare species. We identified a differential effect on phylogenetic groups, with Diversisporales richness mainly reduced by N additions in contrast to Glomerales highly significantly affected solely by P. Regarding AMF community structure, we observed a compositional shift when analyzing presence/absence data following P additions. In conclusion, N and P additions in this ecosystem affect AMF abundance, but especially AMF species richness; these changes might influence plant community composition and productivity and by that various ecosystem processes.

Abstract:

Previous research from the tropics indicates that AMF may be well adapted to organic soils and even represent the dominant mycorrhizal form, though the extraradical part of the symbiosis was omitted as in most other tropical studies. Our study aims at characterizing the extraradical part of arbuscular mycorrhizal fungi (AMF) in a highly organic tropical montane forest soil in Southern Ecuador. Based on recent studies on the interaction of AM fungal hyphae and litter we hypothesized that within the organic layer AM hyphae grow in close contact with decomposing material. To test this idea, AM fungal hyphal distribution in the organic layer was determined by directly staining roots and decomposing leaves and extracting hyphae from the remaining particulate organic material. AM and non-AM fungal hyphae were analyzed, as well as root colonization patterns. Our results showed that AMF indeed represented the dominant mycorrhizal form with an average root colonization of 43%. The extraradical AM hyphal length ranged from 2 to 34 m g?1 soil with a mean of 10.4 m g?1 soil (equals 3.1 m cm?3 soil), and therefore exceeded root length about 13-fold. As hypothesized, 29% of AM extraradical hyphae were closely attached to decomposing leaves. These hyphae were mainly located at the leaf surface, though in some parts leaf veins and inner leaf tissues were colonized. More than half of AM hyphal biomass was detected on the root surface, a pattern potentially driven by the predominant Paris-type AMF. Non-AM fungal hyphae colonized decomposing material to a significantly greater extent, though hyphal length attached to roots was equal. This study supports the adaptation of AMF to highly organic soils in the tropics and the existence of a widespread extraradical mycelium, which is not readily detectable by standard methods. The close association with decomposing leaves most likely improves direct nutrient uptake from decomposed material and points to a potential indirect contribution of AMF to the decomposition process.