Diversity of Marine-Derived Fungal Cultures Exposed by DNA Barcodes: The Algorithm Matters.

Marine fungi are an understudied group of eukaryotic microorganisms characterized by unresolved genealogies and unstable classification. Whereas DNA barcoding via the nuclear ribosomal internal transcribed spacer (ITS) provides a robust and rapid tool for fungal species delineation, accurate classification of fungi is often arduous given the large number of partial or unknown barcodes and misidentified isolates deposited in public databases. This situation is perpetuated by a paucity of cultivable fungal strains available for phylogenetic research linked to these data sets. We analyze ITS barcodes produced from a subsample (290) of 1781 cultured isolates of marine-derived fungi in the Bioresources Library located at the Australian Institute of Marine Science (AIMS). Our analysis revealed high levels of under-explored fungal diversity. The majority of isolates were ascomycetes including representatives of the subclasses Eurotiomycetidae, Hypocreomycetidae, Sordariomycetidae, Pleosporomycetidae, Dothideomycetidae, Xylariomycetidae and Saccharomycetidae. The phylum Basidiomycota was represented by isolates affiliated with the genera Tritirachium and Tilletiopsis. BLAST searches revealed 26 unknown OTUs and 50 isolates corresponding to previously uncultured, unidentified fungal clones. This study makes a significant addition to the availability of barcoded, culturable marine-derived fungi for detailed future genomic and physiological studies. We also demonstrate the influence of commonly used alignment algorithms and genetic distance measures on the accuracy and comparability of estimating Operational Taxonomic Units (OTUs) by the automatic barcode gap finder (ABGD) method. Large scale biodiversity screening programs that combine datasets using algorithmic OTU delineation pipelines need to ensure compatible algorithms have been used because the algorithm matters.

Bacterial community dynamics in the marine sponge Rhopaloeides odorabile under in situ and ex situ cultivation.

Cultivation of sponges is being explored to supply biomaterial for the pharmaceutical and cosmetics industries. This study assesses the impact of various cultivation methods on the microbial community within the sponge Rhopaloeides odorabile during: (1) in situ cultivation under natural environmental conditions, (2) ex situ cultivation in small flow-through aquaria and (3) ex situ cultivation in large mesocosm systems. Principal components analysis of denaturing gradient gel electrophoresis profiles indicated a stable microbial community in sponges cultured in situ (grown in the wild) and in sponges cultured ex situ in small flow-through aquaria over 12 weeks. In contrast, a shift in the microbial community was detected in sponges cultivated ex situ in large mesocosm aquaria for 12 months. This shift included (1) a loss of some stable microbial inhabitants, including members of the Poribacteria, Chloroflexi and Acidobacteria and (2) the addition of new microbes not detected in the wild sponges. Many of these acquired bacteria had highest similarity to known sponge-associated microbes, indicating that the sponge may be capable of actively selecting its microbial community. Alternatively, long-term ex situ cultivation may cause a shift in the dominant microbes that facilitates the growth of the more rare species. The microbial community composition varied between sponges cultivated in mesocosm aquaria with different nutrient concentrations and seawater chemistry, suggesting that these variables play a role in structuring the sponge-associated microbes. The high growth and symbiont stability in R. odorabile cultured in situ confirm that this is the preferred method of aquaculture for this species at this time.

Temperature thresholds for bacterial symbiosis with a sponge.

The impact of elevated seawater temperature on bacterial communities within the marine sponge Rhopaloeides odorabile was assessed. Sponges were exposed to temperatures ranging between 27 and 33 degrees C. No differences in bacterial community composition or sponge health were detected in treatments between 27 and 31 degrees C. In contrast, sponges exposed to 33 degrees C exhibited a complete loss of the primary cultivated symbiont within 24 h and cellular necrosis after 3 days. Furthermore, denaturing gradient gel electrophoresis (DGGE) and clone sequence analysis detected a dramatic shift in bacterial community composition between 31 and 33 degrees C. Within the first 24 h most of the DGGE bands detected in samples from 27 to 31 degrees C were absent from the 33 degrees C sponges whereas eight bands were detected exclusively in the 33 degrees C sponges. The 16S rRNA sequencing revealed that most of the microbes from sponges exposed to 27-31 degrees C had highest homology to known sponge-associated bacteria. In contrast, many of the microbes from sponges exposed to 33 degrees C were similar to sequences previously retrieved from diseased and bleached corals. The 16S rRNA clone library analysis also detected a significant shift in bacterial community structure. The 27 degrees C library was composed of Proteobacteria, Actinobacteria, Nitrospira, Acidobacteria and Chloroflexi whereas the 33 degrees C library contained sequences from the Proteobacteria, Bacteroidetes and Firmicutes. The clear shifts in community composition at elevated temperatures can be attributed to the loss of symbionts and to the establishment of alien microbial populations including potential pathogens. Breakdown of symbioses and stress in the sponge occurred at temperatures identical to those reported for coral bleaching, indicating that sponges may be similarly threatened by climate change.