title: Investigating and comparing chemical and biological variation of marine sponges (phylum Porifera)
Creators: name: Habener, Leesa
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Chemical variation
subject: Biological variation
subject: Marine sponges
subject: HSQC spectroscopic profiling
subject: Spectroscopic profiles

description: The work completed in this PhD thesis investigates and compares the chemical and biological variation of marine sponges. A comprehensive literature review of compounds isolated from the family Mycalidae was completed to characterise the known chemical diversity of the family. In total 190 compounds were reported from 40 taxa comprising a range of structure classes that include alkaloids, polyketides, terpene endoperoxides, peptides, and lipids. The family is identified as a good target for biodiscovery; macrocyclic polyketides display potent cytotoxicity (LC50 &lt;1 μM), and alkaloids (pyrrole derivatives) moderate cytotoxicity (LC50 1 – 20 μM). The pyrrole alkaloids and cyclic peroxides are phylogenetically restricted to sponges and could provide a good candidate for chemotaxonomic markers. Morphological taxonomy of sponges is complicated by a lack of clear diagnostic morphological characters, and chemical compounds are a useful set of characters that can be explored. A 2D 1H-13C HSQC spectroscopic profiling technique was developed to assess the chemical variation of sponge extracts. HSQC spectroscopic profiles (n=28) comprising 6 species of three sponge families (Microcionidae, Mycalidae, and Raspailiidae) showed consistent chemical profiles within family and over geographic scales ranging from approximately 30 to 1700 km. Comparison of chemical shift values to the literature resulted in the identification of chemical compounds that contain chemical moieties indicated by the diagnostic H-C signals. LC-MS/MS enabled identification of 22 known compounds, of which one, mycalamide A, was a sponge natural product. HSQC spectroscopic profiling provides a powerful technique to characterise the chemical moiety diversity that can be applied to both sponge taxonomy and natural products discovery. Molecular approaches have also been applied for the identification of sponge species. Three mitochondrial markers (CO1, SP1, and SP2) were amplified for a collection of sponges (n=144) from the Great Barrier Reef, Australia to group samples into sets that share genetic similarity. This resulted in the establishment 34 molecular operational taxonomic units (MOTUs) and 27 of these MOTUs were assigned order-level identifications using phylogenetic reconstructions. Initially low amplification rates across all three markers (ranging from 17.9 – 34.0 %) were improved after cleaning DNA extracts with phenol-chloroform and reattempting amplification (increased to 60.4 – 75.5 %) for a subset of samples (n=106) indicating the presence of chemical inhibitors. Integrative taxonomic approaches combining multiple datasets have been proposed to assist sponge taxonomy. The developed HSQC spectroscopic profiling approach was applied to a subset (n=56) of the same samples collected from the Great Barrier Reef. Analysis of spectroscopic profiles allowed the identification of three chemical operational taxonomic units (ChOTUs) that displayed high chemical similarity. Searching the literature resulted in the identification of chemical compounds that contain chemical moieties indicated by the distinguishing H-C signals. The morphological operational taxonomic units (OTUs) established at time of collection, and the MOTUs and ChOTUs established here, were used to propose three putative taxa as a first step in species delimitation. Overall this thesis proposes a HSQC spectroscopic profiling method that can be applied to sponge taxonomy as well as to natural products discovery efforts. Spectroscopic profiles can be used to de-replicate samples and target samples possessing unique chemical profiles. Through this process sponge families were shown to display consistent spectroscopic profiles over large geographic scales; an important finding for both sponge chemical taxonomy and biodiscovery projects. Additionally, this thesis results in the proposal of three putative taxa on the basis of comparing chemical, morphological, and molecular variation.