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Biodivers Conserv (2008) 17:27012712 DOI 10.

1007/s10531-007-9247-6 O R I G I NA L P AP E R

Leaf litter fungi in a Central Amazonian forest: the inXuence of rainfall, soil and topography on the distribution of fruiting bodies
Ricardo Braga-Neto Regina Celi Costa Luizo William Ernest Magnusson Gabriela Zuquim Carolina Volkmer de Castilho

Received: 5 April 2007 / Accepted: 24 August 2007 / Published online: 15 September 2007 Springer Science+Business Media B.V. 2007

Abstract Fungi are important components of tropical ecosystems, especially in the recycling of nutrients. However, there is little information on how fungal diversity is structured at scales suitable to plan their conservation. We tested if the distribution of fruiting bodies of litter fungi was random in the landscape (over 25 km2) in a tropical evergreen forest in Central Amazonia. We used linear regressions to evaluate the inXuence of rainfall, soil characteristics and topography on morphospecies richness and composition. Fungi were collected twice in thirty 0.25 250 m plots. Short-term rainfall was represented by the cumulative rainfall in the three days before each plot was surveyed. Plots were classiWed in two groups based on cumulative rainfall. Clay content in soil and rainfall inXuenced morphospecies richness, but responses to edaphic factors depended on rainfall. Wetter periods apparently decreased limiting moisture conditions in higher areas, allowing fungal activity and fruiting body production. Morphospecies composition was inXuenced by clay content, but inXuence on fungi was probably indirect as clay content was correlated with altitude, plant community and nitrogen availability. Our results suggest that the species of litter fungi are not randomly distributed in the landscape. Furthermore, they indicate that it is viable to conduct mesoscale evaluations of fungal diversity, if the temporal and spatial variation and their interaction are taken into account. Resumo (in Portuguese) Fungos so importantes componentes dos ecossistemas tropicais, atuando especialmente na reciclagem de nutrientes. Entretanto, existe pouca informao sobre como a diversidade de fungos est estruturada em escalas adequadas para planejar sua conservao. Ns testamos se a distribuio de corpos de frutiWcao de fungos de liteira ocorre de forma aleatria na paisagem (em 25 km2) em uma Xoresta na Amaznia Central. Utilizamos regresses lineares para avaliar a inXuncia da precipitao, do solo e topograWa sobre a riqueza e composio de morfoespcies. Os fungos foram

R. Braga-Neto (&) R. C. C. Luizo W. E. Magnusson G. Zuquim C. V. de Castilho Instituto Nacional de Pesquisas da Amaznia, Coordenao de Pesquisas em Ecologia, Avenida EWgnio Sales, 2239, Manaus, CP 478, CEP 69011970, Brazil e-mail: saci@inpa.gov.br

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amostrados em trinta parcelas de 0,25 250 m em duas ocasies. A precipitao em curto prazo foi representada pela chuva acumulada em trs dias antes da parcela ser amostrada. As parcelas foram classiWcadas em dois grupos baseados na precipitao acumulada. O contedo de argila no solo e a precipitao inXuenciaram a riqueza de morfoespcies, mas as respostas aos fatores edWcos dependeram da precipitao. Perodos mais chuvosos aparentemente diminuram condies limitantes de umidade nas reas mais elevadas, permitindo a atividade e produo de corpos de frutiWcao pelos fungos. A composio de morfoespcies foi inXuenciada pelo contedo de argila, mas provavelmente a inXuncia sobre os fungos foi indireta, dado que o contedo de argila esteve correlacionado com altitude, comunidade de plantas e disponibilidade de nitrognio. Nossos resultados sugerem que as espcies de fungos de liteira no esto distribudas aleatoriamente na paisagem. Alm disso, indicam que conduzir avaliaes da diversidade de fungos em mesoescala vivel, desde que a variao temporal e espacial, e sua interao, sejam consideradas. Keywords Beta diversity Biodiversity inventories Community ecology Environmental gradients Fruiting body production Litter decomposition Mesoscale Nutrient cycling Patterns of distribution Tropical fungi Abbreviations INPA (Instituto Nacional de Pesquisas da Amaznia) RFAD (Reserva Florestal Adolpho Ducke) RAPELD the Portuguese acronym for National Institute of Amazonian Research the Portuguese acronym for Adolpho Ducke Forest Reserve Term coined by Magnusson et al. (2005) applied to their sampling methodology, useful both for rapid access to biodiversity and long-term research the Portuguese acronym for Long Term Ecological Research Program the Portuguese acronym for Biodiversity Research Program First collection occasion for all plots, from 29 June to 18 September 2005 Second collection occasion for all plots, from 20 September to 22 January 2006 Principal Coordinates Analysis Morphospecies richness, given by the total number of morphospecies collected Morphospecies composition, based on the incidence of morphospecies in each plot the Portuguese acronym for Climate and Water Resources Research Coordination

PELD (Pesquisas Ecolgicas de Longa Durao) PPBio (Programa de Pesquisas em Biodiversidade) COL1 COL2 PCoA MR MC CPCRH (Coordenao de Pesquisas em Clima e Recursos Hdricos)

Introduction Fungi are important components of tropical systems (Hawksworth and Colwell 1992), and are responsible for most nutrient and carbon cycling (Swift 1982). Saprotrophic litter fungi contribute substantially to these processes (Hedger 1985) because Wne litter production

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represents the bulk of the input of biomass to the decomposition system in tropical forests (Luizo 1989). Resource distribution in time and space strongly inXuence the activity of litter fungi, as diVerent species have diVerent degrees of resource selectivity, either taxonselectivity or component-selectivity (Hedger 1985; Rayner et al. 1985). The resource represents both the substratum and the source of organic nutrients for these fungi, and its durability aVects directly mycelial longevity (Rayner et al. 1985). The fast rate of decomposition of Wne litter on the ground in tropical forests (Swift et al. 1979; Hedger 1985) necessitates rapid colonization by fungi for capture of resources and subsequently, rapid exit during the colonization of newer resources. Topography and soil properties inXuence the spatial distribution of plant communities in Central Amazonia (Costa et al. 2005; Kinupp and Magnusson 2005). However, there is little information on how those aVect the distribution of fungi in the landscape. Most species of fungi in the Agaricales are believed to have restricted spatial distributions (Lodge et al. 1995), but owing to the small scale of sampling and the lack of standardized methods among studies, there is still little understanding of how the diversity is spatially organized at diVerent scales. Lodge et al. (1995) concluded that the diversity of saprobic fungi is higher in areas with low latitudes, medium to low altitudes, moderate to high rainfall, high habitat diversity, and to a lesser extent in areas with higher host diversity and resource abundance. These characteristics are widely present in Amazonia, leading to an expectation of high fungal diversity in evergreen tropical forests, independent of the site. However, the scale of analysis of Lodge et al. (1995) was several times greater than scales where conservation decisions usually take place, and it is diYcult to interpolate their conclusions to determine whether the diversity of fungi is non-randomly distributed in the landscape at smaller scales. Also, although mean annual rainfall may be high in Amazonia, lack of rainfall may limit fungus fruiting body production in dryer years, which may occur due to the warming of PaciWc or Atlantic Ocean superWcial waters (Fearnside 2006). Although the occurrence of fruiting bodies does not necessarily reXect the activity and distribution of mycelia (Rayner et al. 1985), the observation and collection of fruiting bodies have been used to determine macrofungal species occurrence in space and time (Vogt et al. 1992; Lodge and Cantrell 1995; Lodge et al. 2004; ODell et al. 2004). However, temporal variation in the detectability of litter fungi sporocarps confuses the perception of spatial variation in the occurrence of species. One of the impediments to landscape studies of fungi is the diYculty of identifying species in the Weld, even when fruiting bodies are present. Microscopic examination is necessary to separate many species, especially those in Tricholomataceae (Singer 1986), and speciWc determination of large collections by specialists may take years or decades. These problems are exacerbated in tropical areas, where many species may be undescribed. However, given the importance of fungi to ecosystem processes, these problems should not be used to exclude fungi from biodiversity surveys. Our preliminary surveys showed that about 70 % of species in a tropical forest in Central Amazonia can be sorted to morphospecies based only on macroscopic characteristics, and these species represent the majority (about 90%) of sporocarps encountered. Currently, these materials are identiWed only by herbarium accession numbers, but a photographic guide to morphospecies (Braga-Neto 2007) permits comparison with other sites. We investigated whether temporal variation in rainfall and spatial variation associated with soil and topography were correlated with the distribution of fruiting bodies of common species of litter decomposer fungi over an area of 25 km2 in evergreen tropical forest in Central Amazonia during a dry year.

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Study site The study was conducted at Reserva Florestal Adolpho Ducke (RFAD), located on the outskirts of Manaus, Brazil (0255 S 5959 W; Fig. 1). The reserve protects about 10,000 ha (10 10 km) of terra Wrme forests, but is in contact with urban areas on the southern and western borders. The highly leached RFAD soils belong to the Alter do Cho geological formation (Chauvel et al. 1987; Sombroek 2000). The topography is variable, intermingling ridge tops, slopes and lowlands, which may be Xooded partially after intense rainfall, especially during the wet season. The altitude varies from 40 to 110 m above sea level. In RFAD, variations of soil texture are strongly associated with topography (Chauvel et al. 1987; Mertens 2004). Rainfall is concentrated between November and June, with highest precipitation in March and April. There are about three months with less than 100 mm of rainfall, between July and September (Marques Filho et al. 1981). The annual rainfall is 2436 332 mm (mean standard deviation), with amplitude of 13002900 mm (from 1975 to 2004). Mean temperature is about 26C, and varies little through the year. The dry season in 2005 had little rainfall due to the warming of Atlantic Ocean superWcial waters (Fearnside 2006; Trenberth and Shea 2006).

Methods Sampling design Following the RAPELD methodology (Magnusson et al. 2005), we used thirty 0.25 250 m plots, maintained by the Brazilian Long Term Ecological Research Program (PELD) and the Program for Biodiversity Research (PPBio) at RFAD. Plots are orientated along the topographic contours, which reduces the variation in factors correlated with altitude, such as soils (Chauvel et al. 1987; Mertens 2004). The 30 plots are distributed on a grid system of 5 5 km, and plots were at least one kilometer apart. The utilization of long thin plots increases the number of species registered in comparison with square or circular plots of the same area (Krebs 1998; Cantrell 2004).

Fig. 1 Location of Reserva Florestal Adolpho Ducke (RFAD, 0255 S 5959 W) and the grid system inside the reserve. Dots on the trail system indicate the location of permanent plots. Lines represent topographic contours, ranging from 40 to 110 m above sea level; darker lines indicate higher areas

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Data collection Fungi The fungi were collected between June 2005 and January 2006. Each of the 30 plots was visited twice. The Wrst collection (COL1) occurred from 29 June to 18 September 2005, and the second (COL2), from 20 September to 22 January 2006. The number of days between sampling occasions for each plot was 109 17.3 (mean standard deviation). Marasmioid, crepidotoid and collybioid fungi connected to Wne litter components were sampled. Only fruiting bodies visible at the surface were collected; the litter was not turned over. The specimens were photographed in situ whenever feasible. Fruiting bodies were measured and described macroscopically, and subsequently dried. For most specimens, photographs and macroscopic data were available for each sample. Specimens were classiWed into morphospecies. The descriptions were used to construct a preliminary guide to morphospecies of litter fungi at RFAD (Braga-Neto 2007). Some species of fungi cannot be distinguished macroscopically, and only data for morphospecies that could be consistently grouped on macroscopic characters (about 70%) were used in the analyses presented here. All fruiting bodies collected were deposited in the INPA Herbarium. Environmental variables Rainfall data were collected at the RFAD Meteorological Station, which is located outside of the grid system, between June 2005 and January 2006. Altitude and soil variables data were obtained from the PPBio database. Variations in soil texture are strongly associated with topography in the study region (Chauvel et al. 1987). Within the RFAD, as elsewhere in Central Amazonia (Luizo et al. 2004), clay content was strongly correlated with altitude (r = 0.977) and sand content (r = 0.999), such that clay predominated in higher areas and sand in the lowlands. Moreover, clay content predicted much of the spatial variation in the communities of herbs (Costa et al. 2005) and shrubs (Kinupp and Magnusson 2005). Thus, clay content was chosen to represent the correlated edaphic variables, but the inXuence on fungi may be indirect. Data analysis We tested possible correlation in fungus composition between the two sampling periods and spatial auto-correlation of morphospecies richness and composition (Legendre and Legendre 1998). Mantel tests were used to evaluate spatial auto-correlation, using the Euclidean distance for richness and spatial distance and the Srensen index for community composition (incidence data). Additionally, we tested the spatial auto-correlation of clay content in soil using the Euclidean distances of standardized clay data against the spatial distance. Rainfall was represented by the cumulative rainfall in the three days before each plot was surveyed, a period assumed to allow fruit body production in response to rainfall. Plots were grouped in two rainfall categories. The plots with more than 10 mm of three-day cumulative rainfall were considered as high-rainfall collection plots (N = 15) and the ones sampled after a three-day period in which cumulative rainfall was less than 10 mm were considered as low-rainfall collection plots (N = 45). No plot classiWed in the high-rainfall group was repeated. However, in the low-rainfall group 15 plots were sampled twice, and one of the sampling occasions was randomly excluded for each of these plots to avoid temporal pseudoreplication. Hence, the same plot was not counted twice in either treatment.

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Richness (number of morphospecies) and morphospecies composition were used in diVerent analyses to investigate the distribution of the litter fungus community. The multidimensionality of the community composition was reduced by principal coordinates analysis (PCoA) based on incidence data (presence or absence of morphospecies in each plot), using the Srensen index as a community composition distance measure. Gotelli and Ellison (2004) recommend the use of PCoA when the objective is to preserve the original multivariate distances between observations in the reduced space, especially in analyses of presence-absence data. The cumulative variance explained by the Wrst three axes was about 51% for plots surveyed in low-rainfall periods, and about 57% for plots surveyed in high-rainfall periods. We excluded three plots with zero morphospecies in the composition analysis. The morphospecies richness and the three ordination axes resulting from PCoA, which describe morphospecies composition, were used as dependent variables in inferential tests of the eVects of soil, topography and rainfall. Multiple regressions were used to analyze relationships between variables, presuming that distinct predictor variables cause an eVect in the response variable. Linear regression is the basis of most multivariate models (McCune and Grace 2002). Hence, the predictive value of environmental variables was evaluated with multiple linear regression analysis for morphospecies richness and with multivariate multiple regressions for morphospecies composition. The environmental model tested included the rainfall estimate (three-day cumulative) and soil clay content. The estimate of rainfall was transformed to natural log to achieve linearity. Tolerances for all variables in all models were higher than 0.3. The Pillai Trace multivariate statistic was used to determine the signiWcance of each variable and for the whole model. Outliers were excluded when necessary. Two plots were outliers in all analyses. They were sampled in the Wrst day of the study in June 2005, after a short period with little rainfall immediately following the wet season (Fig. 2), and probably represent relationships distinct from those that prevailed during the rest of the study. Therefore, we present results including and excluding those plots. Ordinations were done with the PATN package (Belbin 1992), Mantel tests with the RT package (Manly 1997) and the regression analyses were performed in Systat 8.0 (Wilkinson 1998).
180 30 25 20 15 10 5 0 COL1 COL2

160 140 120 100 80 60 40 20 0

Fig. 2 Distribution of values of the three-day cumulative rainfall estimate (bars) and cumulative number of sampled plots (dots) for each sampling occasion within each collecting period in this study in RFAD, Brazil. The two sampling occasions (COL1, from 29 June to 18 September 2005 and COL2, from 20 September to 22 January 2006) are indicated

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Results Sampling eVort and rainfall pattern Most plots were sampled in the dry and early wet seasons of 2005 (Fig. 2). A total of 1,193 fruit bodies were recorded and classiWed into 87 morphospecies. During the Wrst sampling period (COL1), 635 fruit bodies were assigned to 61 morphospecies. In the second period (COL2), 558 fruit bodies were classiWed into 58 morphospecies. There was no diVerence among sampling periods for the observed number of morphospecies (t = 1.290, P = 0.203) or the number of fruiting bodies (t = 0.363, P = 0.718). Only 32 morphospecies were found in both sampling periods. Morphospecies richness was not correlated among sampling occasions (r = 0.061, P = 0.747), nor was it spatially auto-correlated. Morphospecies composition was not spatially auto-correlated, but it was weakly correlated between sampling occasions (r = 0.176, P = 0.001), implying non-independence in the detection of fruiting bodies of most morphospecies between sampling occasions. Mantel tests revealed spatial auto-correlation for clay content (r = 0.115, P = 0.017) when all 30 plots are considered in the low-rainfall group, but the relationship is so weak that it does not inXuence the interpretation of results. Responses of litter fungi community to rainfall and clay content For collections made in the low-rainfall periods (three-day cumulative rainfall <10 mm), morphospecies richness (MR) was related to the predictor variables (MR = constant + rainfall + clay, F2, 24 = 8.250, P = 0.002, R2 = 0.358, N = 27); both rainfall (P = 0.002) and clay content (P = 0.040) contributed signiWcantly to the model. Fewer morphospecies were found in dry periods with less rainfall (Fig. 3a) and in collections on clay soils (Fig. 3b). For collections made in the high-rainfall periods (three-day cumulative rainfall > 10 mm), MR was also signiW cantly inXuenced by the model (MR = constant + rainfall + clay, F2, 11 = 4.456, P = 0.038, R2 = 0.347, N = 14). Rainfall (P = 0.046) contributed signiWcantly to the model (Fig. 3c), but clay content did not (P = 0.198, Fig. 3d). Clay content did not contribute signiWcantly to the model (P = 0.076) for low-rainfall group when all the plots, which were outliers were included (MR = constant + rainfall + clay, F2, 27 = 3.129, P = 0.060, R2 = 0.128, N = 30). The outlying plots all had much higher morphospecies richness than expected for plots collected in low-rainfall periods. Two were sampled immediately following the rainy season, which generally ends in MayJune in the region, so soil and leaf-litter water potentials were still high, despite the lack of rainfall in the previous three days. Excluding these plots, the relationship between clay content and morphospecies richness was signiWcant for plots collected in the low-rainfall periods. For collections made in low-rainfall periods, morphospecies composition (MC) was inXuenced by the model (MC = constant + rainfall + clay, F6, 44 = 3.699, P = 0.005, R2 = 0.267, N = 26), but only clay content (P < 0.001) contributed to the model; there was no evidence for an eVect of rainfall (P = 0.678). There was also some evidence for an eVect of the variables in collections in high-rainfall periods (MC = constant + rainfall + clay, F6, 2 22 = 2.474, P = 0.056, R = 0.301, N = 15) and the pattern was similar to that in the dry periods, with low probability for the null hypothesis associated with clay (P = 0.051) and no evidence for an eVect of rainfall (P = 0.223).

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4 3 2 1 0 -1 -2 -3 -4 3 2 1 0 -1

Morphospecies Richness (partial)

Low-rainfall Period (< 10 mm)

1 0 -1 -2 -3 -1.0 -0.5 0.0 3 2 1 0 -1 -2 -3 -4 -5 -1.0 0.5 1.0

a
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0 0 0 0 0 -5 -4 -3 -2 -1 0 10 20 30 40 50

High-rainfall Period (> 10 mm)

c
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-2 -3

d
Clay Content (partial)

Log(Rainfall) (partial)

0 0 0 0 0 -5 -4 -3 -2 -1 0 10 20 30 40 50

Fig. 3 Partial plots of the eVects of rainfall and clay content on morphospecies richness for low-rainfall (three-day cumulative rainfall <10 mm) periods (a, b), and for high-rainfall (three-day cumulative rainfall > 10 mm) periods (c, d)

Discussion The community of litter fungi seemed to be spatially structured over 25 km2 along rainfall and edaphic gradients. Our results indicated that litter fungal fruiting body production in a tropical forest varied predictably in space and time, suggesting that litter fungi species are not randomly distributed at mesoscales. Rainfall and clay content in soil predict morphospecies richness and composition, but responses of the number of morphospecies to edaphic factors depended on rainfall. Relationships between morphospecies richness and soil texture were not constant along the rainfall gradient. Clay content, which was correlated with topographic position, inXuenced morphospecies richness only in low-rainfall periods, while in the high-rainfall periods richness was not related to soil characteristics. Rainfall is a primary determinant of the availability of water in forest ecosystems, but the gradient of moisture in the RFAD landscape seemed to depend on drainage patterns, which is mainly determined by topography. Low clay content is associated with lowlands and proximity to watercourses in the study site (Chauvel et al. 1987). These areas are relatively wetter, especially in the driest months of the year. This trend may favor mycelial activity, fruiting body production, and the chance of observation of species. The absorptive nutrition of fungi implies dependence on substrate moisture conditions. Daily and seasonal variances are determinants of litter fungi mycelial activity and fruiting body production in tropical forests (Hedger 1985). Though optimal moisture conditions vary among species, litter fungal fruiting body production is expected to be lower in periods with low humidity (Singer and Araujo 1979). The absence of a signiWcant correlation between the number of morphospecies and clay content for the high-rainfall periods may be related to a decrease in topographic inXuence

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over the moisture gradient, presumably caused by less limiting moisture conditions driven by higher rainfall. On the other hand, rainfall inXuences on morphospecies richness were consistent among rainfall groups. We found an expected positive relation between morphospecies richness and three-day cumulative rainfall for both rainfall groups, since litter fungal fruit body production depends on short-term rainfall (Singer and Araujo 1979). However, the magnitude of the eVect is likely to be related to the fact that we sampled during a relative dry year, and probably the eVects are weaker in wetter years. Soil texture inXuences on litter fungus community composition Morphospecies composition was inXuenced by clay content in soil within the low-rainfall group, and approached signiWcance for the high-rainfall plots. These results suggest that fruit body production by the litter fungus community is spatially structured along edaphic gradients over 25 km2, although there is no evidence of these eVects on the mycelial distribution. Given the strong correlation with altitude (Chauvel et al. 1987), clay content seems to reXect the landscape moisture gradient, hence inXuencing the distribution of litter fungus morphospecies, as species are supposed to diVer in their abilities to deal with moisture (Hedger 1985). Moreover, clay content inXuences plant community spatial distribution (Costa et al. 2005; Kinupp and Magnusson 2005), is positively correlated with leaf litter N concentrations (Luizo et al. 2004) and is supposed to be negatively associated with ectomycorrhizal fungi (Singer 1978; Singer and Araujo 1979) in Central Amazonia. The inXuence of plant community composition on the spatial distribution of litter fungi is often considered to be high, due to taxon selectivity (Singer 1976; Cornejo et al. 1994; Lodge et al. 1995; Polishook et al. 1996), although quantitative data on this issue for basidiomycete fungi are scarce. Santana et al. (2005) found that matching of decomposer fungi to the leaf substratum by the host plant family or chemical/physical traits of the source plant in which they were dominants increased rates of decomposition. At large scales, spatial distribution patterns of Marasmius spp. and Marasmiellus spp. fungi do coincide with patterns of phanerogam plants (Redhead 1989). In RFAD, plants are not randomly distributed in space, but spatially structured by topographic and edaphic factors (Costa et al. 2005; Kinupp and Magnusson 2005). As litter fungal species possess some degree of taxon selectivity (Singer 1976), one expects that they are inXuenced by the distribution of their hosts. Additionally, litter fungi species are expected to have diVerent demands for resource nutrient composition (Hedger 1985). The litter layer N concentration may be lower in the valley plots, where soils have low clay content, as elsewhere in Central Amazonia (Luizo et al. 2004). However, leaf litter nutrient concentrations may be related to soil nutrient status rather than topography (Wood et al. 2006). In a nearby site, Singer and Araujo (1979) observed more species of litter fungi on clay soils, attributing the pattern to negative inXuence of competitive ectomycorrhizal fungi in sandy soils (Singer 1978). However, we found the opposite pattern. The number of morphospecies of litter fungi detected was higher in sandy sites. Besides the diYculties in the comparison of results due to diVerent sampling designs, drier environmental conditions may have limited the detectability of litter fungi species in sites with higher clay content during our study. The correlation of morphospecies composition between sampling occasions may reXect seasonality in fruiting body production, and implies non-independence in the detection of fruiting bodies of most morphospecies between sampling occasions. Non-independence is a problem with repeated measures analysis, because time series are likely to be correlated, such that current values are a function of values observed in the past (Gotelli and Ellison

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2004). However, temporal correlation between samples does not mean one cannot incorporate time series data into conventional statistical analyses. Temporal replicate sampling is very appropriate to study how ecological systems change trough time, and many conservation models designed to predict future conditions are derived more reliably from replicated data in time (Ferraz et al. 2007). Some alternatives to repeated measures analysis may be especially useful to avoid pseudoreplication problems (Gotelli and Ellison 2004). Our approach to avoid problems related to lack of independence of the data and to maximize data use was to classify all plots collected in two groups (based on 10 mm of three-day cumulative rainfall), not to count the plot twice in either group, and to analyze the data separately. The further apart in time the samples are separated from one another the more likely they function as independent replicates (Gotelli and Ellison 2004). In our study, the number of days between the sampling occasions for each plot was similar, although it was not suYcient to guarantee independence. Future sampling designs on leaf litter fungus should consider the interval between samples of this study (about four months), aiming to avoid temporal correlation among samples. This information may be extremely useful for biodiversity inventory planning, as it may be possible to take into account diVerent species detectabilities while reducing sampling costs in time and Wnancial resources. Caveats and implications for fungal conservation Although no diVerences in abundance or morphospecies richness between the Wrst and second sampling periods were found, the low proportion of shared morphospecies suggests that an increase in rainfall in the early wet season of 2005 triggered fruiting body production of diVerent species, an indication of diVerential responses to seasonal/moisture regimes. However, most morphospecies were infrequent or rare, and the detectability of most litter fungal species seems to be low. Repeated surveys of the same plots will be needed to detect most species. This study has shown that rainfall, probably through its eVect on litter water potential, can strongly aVect apparent habitat speciWcity in fungi. Although we showed only temporal eVects associated with an uncommonly dry year (Fearnside 2006), spatial eVects in areas with lower rainfall, typical of much of the Amazon basin, are likely. Therefore, it is important that studies of spatial variation in fruiting body production take into account the temporal variation caused by weather. However, fungi are likely to be responding to water potential in the litter rather than rainfall per se. The distribution of plots in mesoscale (25 km2) incorporates more environmental heterogeneity than sampling at smaller scales and it is important to improve the quality of moisture estimates in space, either by directly measuring moisture in leaf litter or by including more rainfall collecting stations within the grid system. SigniWcant eVorts have been made to improve global fungal diversity estimates (Hawksworth 1991, 2001), incorporating available data on biogeographic distributions, levels of endemism and host speciWcity (Schmit et al. 2005; Mueller et al. 2006; Schmit and Mueller 2006). However, scarcity of available information in tropical regions, especially in Amazonia, will preclude the test of these estimates and fungal conservation planning as long as large-scale inventories are not accomplished. Most of the unknown fungal diversity is expected to occur in the tropics (Lodge et al. 1995; Hawksworth and Rossman 1997; Hawksworth 2001), and most species distribution ranges are believed to be small (Lodge et al. 1995; Mueller et al. 2006). Lack of methodological standardization between studies restricts the understanding of fungal diversity spatial structure at scales important for

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biological conservation. Both large and small scale datasets are needed to assess fungal species spatial distributions and levels of endemism (Mueller et al. 2006). Our results show that it is viable to conduct mesoscale evaluations of fungal diversity, as long as the temporal and spatial variation and their interaction are taken into account.
Acknowledgements Ricardo Braga-Neto acknowledges the support of a Brazilian National Research Council (CNPq) graduate scholarship. Brazilian Long Term Ecological Research (PELD) guaranteed Weld support and equipment acquisition. The European Network for Research in Global Change (ENRICH) provided support during the preparation of this manuscript. Topographic and edaphic data were obtained from PELD and PPBio (Programa de Pesquisas em Biodiversidade). Eleusa Barros, Tnia Pimentel and Jane Mertens contributed to soil data acquisition. Rainfall data were obtained from CPCRH/INPA (Coordenao de Pesquisas em Clima e Recursos Hdricos). We thank Dennis Desjardin, Carla Puccinelli and Dbora Drucker for their contributions; and D. Jean Lodge, Roberto Garibay Orijel and two anonymous reviewers for revising earlier versions of the manuscript.

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