Abstract:We present the first characterization of fungal community diversity of natural mixed-species biofilms on artificial marine reefs. Four artificial reefs in the Mississippi (MS) Sound, USA, representing low-profile (underwater) and high-profile (periodically air-exposed) conditions were sampled every 3 months over a 23-month period to investigate changes in fungal diversity within reef biofilms. Fungal presence was assessed via PCR amplification of the internal transcribed spacer (ITS) region of fungal ribosomal DNA, and by terminal restriction fragment length polymorphism (T-RFLP) analysis of fungal ITS regions -the latter being used to track variation in fungal community structure with respect to season, location, and reef profile type. Fungal communities were also characterized taxonomically through both morphological identification and phylogenetic comparisons of ITS gene sequences, with 36 fungal genera cultured from reef biofilms. Using a multivariate statistical approach, significant temporal and spatial differences in fungal biofilm communities were detected. High-profile reefs differed significantly in biofilm fungal community composition across the 10 sampling periods. This assessment of marine fungal biofilm communities over time provides novel insights into the fungal diversity present on artificial reefs in an understudied region, the north-central Gulf of Mexico.