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Microbial mats are a laminated organic-sedimentary ecosystem, found in a wide range of habitats. Fluctuating diel and seasonal physicochemical gradients characterize these ecosystems, resulting in both strata and microenvironments that harbor specific microbial communities. This study was undertaken to compare two types of microbial mats across seasons to further understand the structure of fungal communities in hypersaline microbial mats and their seasonal dynamics. The structure and diversity of fungal communities was documented in young transient and mature hypersaline microbial mats from a tropical region (Puerto Rico) using one culture-dependent and three culture-independent molecular techniques based on the internal transcribed spacer (ITS) region of ribosomal DNA: terminal restriction fragment length polymorphism (TRFLP), denaturing gradient gel electrophoresis (DGGE) and clone libraries. Two microbial mats (one young and transient, one mature) were sampled in Nov 2007 (wet season), Jan 2008 (intermediate season) and Mar 2008 (dry season) in the Cabo Rojo Solar Salterns on the southwestern coast of Puerto Rico. Traditional and molecular techniques revealed strong spatial and temporal heterogeneities in both microbial mats. Higher abundance of isolates and phylotypes were observed during the wet season, and diversity decreased from the top (oxic) to the bottom (anoxic) layers in both seasons. Some of the species isolated belong to the genera Aspergillus, Cladosporium, Hortaea, Pichia and Wallemia, which often are isolated from hypersaline environments. The most abundant clones belong to Acremonium strictum and Cladosporium halotolerans, which were not isolated in pure culture. The differences observed using culture-based and molecular techniques demonstrates the need of combining methods to study the diversity of fungi in a given substrate.