Anais da Academia Brasileira de Ciências (2018) 90(2 Suppl. 1): 2359-2373 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201720170120 www.scielo.br/aabc | www.fb.com/aabcjournal Assessment of the occurrence and richness of arbuscular mycorrhizal fungal spores by direct analysis of field samples and trap culture – a comparative study PATRÍCIA L. LEAL1,2, TEOTONIO S. DE CARVALHO1, JOSÉ OSWALDO SIQUEIRA1,3 and FATIMA M.S. MOREIRA1 1Departamento de Ciência do Solo, Universidade Federal de Lavras, Av. Doutor Sylvio Menicucci, 1001, Bairro Kennedy, 37200-000 Lavras, MG, Brazil 2Instituto Multidisciplinar em Saúde, Universidade Federal da Bahia, Rua Rio de Contas, 58, Quadra 17, Lote 58, Bairro Candeias, 45029-094 Vitória da Conquista, BA, Brazil 3Instituto Tecnológico Vale para Desenvolvimento Sustentável, Rua Boaventura da Silva, 955, Nazaré, 66055-090 Belém, PA, Brazil Manuscript received on February 20, 2017; accepted for publication on April 6, 2017 ABSTRACT In this work, we hypothesized that two spore-based methods, direct analysis of field samples and trap cultures, simultaneously used for assessment of occurrence and species richness of arbuscular mycorrhizal fungi (AMF) may vary in their efficiency according to the environmental conditions and the total AMF species richness of the evaluated ecosystem. The performance of both methods was analyzed based on two datasets: 1) a complete site x species matrix compiled from two studies in different land uses in the Amazon using direct analysis of field samples and trap cultures. 2) Total number of AMF morphotypes detected by both methods in published manuscripts across several ecosystems. From dataset 1, direct analysis of field samples revealed 57 morphotypes, whereas only 21 of these were detected by trap culture. Community variation (beta diversity) analysis revealed that field samples are far more sensitive in detecting shifts in AMF community composition among land uses than trap cultures in the Amazon region, with the combined results of both methods being not better than that obtained only by direct analysis of field samples. Analysis of dataset 2 showed that the relative performance of trap cultures, using direct analysis of field sample as reference, was inversely related to the total observed AMF species richness. Key words: alpha and beta diversity, diversity assessment methods, Glomeromycota, spore counts, taxonomy. INTRODUCTION form with the vast majority of plant species occurring in most terrestrial ecosystems and The highly specialized Glomeromycotan because of their functional role in plant nutrition, arbuscular mycorrhizal fungi (AMF) are important growth, and community structure. Their presence components of the belowground biodiversity because of the ubiquitous root symbiosis they is usually assessed by quantitative and qualitative assessments of mycorrhizal colonization and by Correspondence to: Fatima Maria de Souza Moreira E-mail: [email protected] spore counts and identification from field soils. * Contribution to the centenary of the Brazilian Academy of The latter method provides the baseline to assess Sciences. species richness, abundance, frequency, and to An Acad Bras Cienc (2018) 90 (2 Suppl. 1) 2360 PATRÍCIA L. LEAL et al. draw inferences about community composition. are frequently used together in the hope that they Taxonomic classification of AMF spores is usually can give more accurate assessments of AMF evaluated by the analysis of morphological communities, but this implies in at least doubling characters of AMF asexual spores (Schenck the total costs of the analysis, imposing restrictions and Pérez 1988) and by molecular techniques on the sampling effort under a limited availability targeting specific rRNA gene sequences (Liang of resources. Furthermore, the extent to which the et al. 2008). Although suitable protocols and complementarity between these methods is true primers (Helgason et al. 1998, Kruüger et al. and its dependency on environmental conditions 2012) have been developed to identify AMF have not yet been explicitly addressed. Studies species from field samples (Avio et al. 2009, Öpik have demonstrated contrasting results on AMF et al. 2013), the use of molecular techniques is richness obtained through spore analysis collected still limited because it yields DNA sequences from field versus trap cultures (Stürmer 1998, that may or may not match traditional AMF Ferrol et al. 2004, Tchabi et al. 2008, Robinson- morphospecies (Robinson-Boyer et al. 2009, Young 2012). Extraction and identification of Boyer et al. 2009). Some studies recovered none spores either directly from field soil or from trap or only few species exclusively in trap cultures cultures are commonly used procedures in AMF (Bever et al. 1996, Melloni et al. 2003, Oehl et al. ecological and applied research because spores 2003) while others had detected a large number of are the ultimate fungal unit required to establish species (Stutz and Morton 1996, Beauchamp et al. AMF in single cultures, which are then used for 2006). further evaluation of functional traits. Common Thus, mycorrhizal researchers aiming to constraints attributed to field collected spores are evaluate AMF diversity within ecosystems face a the low quantity of spores, parasitism by soil fauna challenge in selecting the method to reveal AMF and bacteria, and lack of all spores subcellular richness and abundance because little data is structures necessary to an accurate identification of currently available with regard to the factors that species. Moreover, not all AMF in the community control the performance of each method and the might be sporulating at the sampling time, what extent to which these methods complement one may underestimate diversity (Morton et al. 1995). another for giving a more accurate picture of AMF Therefore, multiplication of the indigenous fungi communities. In this study, we tested the following in host plants under controlled conditions is an hypotheses: (i) trap culture and direct analysis of alternative for triggering sporulation of cryptic field samples differ in their selectivity for AMF AMF species. This procedure is known as trap species, resulting in method-specific biases on the culture and has been widely used in AMF ecological community composition; (ii) the combination of studies (Bever et al. 1996, Stutz and Morton 1996, Stürmer 1998, Leal et al. 2009, Mergulhão et al. both methods gives a less biased assessment of AMF 2009). It usually gives large number of viable communities and a greater resolution for detecting spores containing all morphological structures for variations in AMF community composition (beta an accurate identification and detection of non- diversity) in response to environmental changes; sporulating AMF species. (iii) the relative performance of trap cultures These two spore-based methods (spores as compared to direct analysis of field samples extracted directly from field soil and trap is variable, depending on specific ecosystem’s cultures) are believed to be complementary and features, such as total richness. An Acad Bras Cienc (2018) 90 (2 Suppl. 1) ARBUSCULAR MYCORRHIZAL FUNGI DIVERSITY METHODS 2361 MATERIALS AND METHODS trap/field and the equivalent percentage. In order to DATASETS calculate the extrapolated species richness for each method, we applied the first-order Jackknife index, Two datasets were used to assess the relative using the package Vegan in the R environment performance of the trap culture method (herein (R Development Core Team 2015). Confidence referred as trap) compared to field spore extraction intervals were calculated from 1,000 resampling (herein referred as field) in recovering AMF cryptic data. species. The first dataset is from of the project To evaluate the association between AMF Conservation and Sustainable Management of spores richness and diversity assessment method Below-Ground Biodiversity, implemented through (trap and field), we used the A component of the the Global Environment Facility. Data on AMF equation for the indicator value index (IndVal) occurrence, abundance and species richness were described by Dufrêne and Legendre (1997), for recovered from field-collected spores (Stürmer and absence and presence data, as follows: Siqueira 2011) and from trap cultures established with the same soil samples from field (Leal et al. √(Apa) = √(np / n) 2009). Both studies include information on AMF Where n is the number of times that a diversity from soils collected at the same time, in particular species occurs across all points; and n the same sampling points from different land uses p is the number of times that the same species occurs (pristine Amazon forest, old secondary forest, for a given group (sampling method). Only non young secondary forest, agroforest systems, field rare AMF species (i.e., those recovered from more crops and pasture) in the western Amazon region, Brazil. Detailed characterization of this area is than two samples) were selected for this analysis. found in Moreira et al. (2009), and methodologies This analysis was performed using the Indicspecies for spore extraction, counts and identification, as package in the R environment (R Development well as trap cultures conditions are described in Core Team 2015). Stürmer and Siqueira (2011) and Leal et al. (2009). For the Amazon dataset, in order to compare The second dataset was compiled from a the community composition indicated by the