New coral associated fungi found at Curacao Master thesis (41.5 ECT’s) Rosan Rongen MSc student Marine Sciences, Utrecht University 26 January January 2018 2 CORAL ASSOSIATED FUNGI Acknowledgement This thesis is written as part for of my master’s programme Marine Sciences at the Utrecht University. The thesis was combined with a practical project at the Microbiology fungal group, from the Biology department at Utrecht University. I greatly enjoyed setting up the idea of this project, carrying out my own ideas and combining this with previous research in this written thesis. This resulted in an enjoyable learning experience. I could not do this without the trust and help from a lot of people. The idea started last year in Curacao, where I did a course for my Master program, I would like to thank Mark Vermeij for this. I got an idea to grow found coral fungi during a lecture of Mark Vermeij, a coral specialist from Carmabi research station. Later he helped me with collecting and sending coral samples to the Netherlands. I would like to thank Han Wösten, from UU, who provided me everything I needed in his lab, his comments during the project, and above all for the useful feedback of this written part. For all practical help in the lab and explanations of methods I would like to thank Martin Tegelaar, from UU. He also provided me with skilful information and ideas when I needed them. Last but not least, I would like to thank my supervisor Jack Middelburg, from UU, who made this project possible for me as an earth sciences student to do research in the biology department. I am grateful for all help and trust above persons gave me to setting up this project. I could not have done it without them. 3 CORAL ASSOSIATED FUNGI New coral associated fungi found at Curacao Thesis written by: Msc student R.M. Rongen Supervisors Prof dr J.B.M. Middelburg Prof dr H.A.B. Wösten PhD student M. Tegelaar A B S T R A C T One of the most biologically diverse ecosystems on earth are tropical coral reefs. It is the habitat for one-third of the marine fish species for at least part of their life cycle. Hard corals are the major structural builders of this system. Nowadays, knowledge of the microbiome of corals is mainly focused on bacteria and symbiotic zooxanthellae. Thus, the importance of fungal coral interactions remains unrevealed which may be both beneficial and pathogenic. Only recently more marine fungi have been studied due to an increase in corals affected by pathogenic fungi. We extended these studies by cultivating endolithic fungi from healthy and diseased coral Sidrastrea siderea, as well as healthy corals of the species Agaricia agaricites, Montastraea annularis, Madracis mirabilis, Stephanocoenia intersepta and Montastraea cavernosa from the reef of Curacao in the Caribbean Sea. Sterilisation by UV light turned out to be a successful method to kill microorganisms at the surface of the coral without affecting the fungi from the internal part. A total of 90 colonies originating from internal parts of corals were purified on plates based on visual inspection, while 4 species were found based on DNA sequencing. Newly found coral associated fungi were Aspergillus tamarii, Penicillium solitum, Aspergillus flavus or Aspergillus oryzae, Aspergillus tubingensis, Aspergillus penicillioides, Penicillium citrinum, and Penicillium westlingii. Aspergillus versicolor, Aspergillus sydowii, were identified in this study as well as in previous studies. Notably, a higher diversity of fungi was cultured from healthy than from diseased S. siderea coral. The identified fungi and what is known from these species suggest that coral associated fungi act as pathogens, bio-eroders, bio-borers, but may also be beneficial as being part of the coral microbiome. In the latter case it may produce mycotoxins, antibacterial, and / or fungal metabolites to protect the coral and its microbiome. In future studies, it would be interesting to combine metagenomics with the approach that was adopted in this study, especially since fungi play a bigger role in coral ecosystems than assumed so far. Introduction One of the most biologically diverse ecosystems on earth are tropical coral reefs. It includes for instance hard and soft-corals, sponges, crustaceans, worms, fish, bacteria, viruses, and fungi. They compete for resources such as food, space and sunlight. Most of the tropical coral reefs have hard corals as the major structural builders of this system1. Natural events such as tsunamis, volcanic eruptions, hurricanes as well as climate change cause shifts in these systems. Climate models predict an increase in the variability of weather; from year to year, and long-term effects, as a result from increasing 2 anthropogenic CO2 concentrations . Tropical coral reef ecosystems are affected by the direct and indirect effect of human population growth3. Protection of coral reefs is not only important from a biodiversity point of view as they are also important for human food supply. 17% Of animal protein originates from fish with about one-third of the marine fish species using coral reefs during their lives4. Other major feedbacks between coral reefs and human kind are pollution and climate change. These proximal drivers have a negative effect on coral reef ecosystems, where ecosystem goods and services as ocean recreation and tourism, and more important coastal protection and food supply will be Figure 1; Feedback of human society and coral reefs. Distal drivers are traits in social systems that indirectly influence how people affected by these proximal drivers (Figure 1)3,5. interact with coral reefs. Proximal drivers directly affect coral reef Corals belong to the Class Anthozoans of the phylum ecosystems (centre). Coral reefs provide important ecosystem services Cnidaria. The largest order is covered by the Scleractinians that to people, which influence human wellbeing. Single-headed arrows consists of the hard corals. Hard corals form the major coral reef indicate how the pathway flows from distal drivers to human structure. Other organisms as crustose coralline algae (CCA), calcium wellbeing. Double-headed arrows show the complex linkages and carbonate forming organisms, and sedimentary fill by all kind of feedbacks that also occur between the various components. Modified organisms contribute too1,6. Hard corals consist mainly of polyps. from Hughes et al. (2017) and Cinner et al. (2015). 4 CORAL ASSOSIATED FUNGI Table 1; Fungi associated with corals (modified from Kendrick et al. (1982) and Raghukumar and Ravindran (2012). Fungi from reference Kendrick et al. (1982) were fungi that were identified after culturing on mycological culture media7,8. * = found in healthy corals. Fungi Coral host Location Reference number Acremonium sp. Meandrina meandrites, Barbados (West Indies), Arabian Sea 7,9 Portites lutea Alternaria sp. Acropora formosa* Great Barrier Reef (GBR) 10 Aspergillus-like Porites lobata, Porites lutea Moorea French Polynesia 11 Aspergillus restrictus Diploastrea heliopora GBR 7 Aspergillus sp. Portites lutea Arabian Sea 9 Aspergillus sydowii Acropora palmata, Montastrea annularis, Barbados (West Indies) 7 Porites porites Aspergillus versicolor Acropora sp., A. hyacinthus, Sidrastrea GBR 7 siderea, Goniastrea retriformis, Diplostrea heliopora, Porites australensis Asteromella sp. Diploastrea heliopora GBR 7 Aureobasidium sp. Portites lutea Arabian Sea 9 Aurobasidium pullulans Acropora formosa GBR 10 Bipolaris rostra Acropora palifera GBR 7 Chaetomium sp. Portites lutea Arabian Sea 9 Cladosporium sp. Portites lutea, Acropora formosa Arabian Sea, GBR 9,10 Cladosporium sphaerospermum Montastrea annularis, Porites sp. Barbados (West Indies), GBR 7 Class: Ascomycetes - Belize (Central America), 8,12 Corallochytrium limacisporum Acropora sp., Porites sp. Arabian Sea 13,14 Fusarium sp. Portites lutea Arabian Sea 9 Genus: Koralionastes - - 15 (5 species not cultured) Hormonema alopallonella A. hyacinthus GBR 7 Hormonema dematioides Acropora sp., Goniastrea retiformis, Porites GBR 7 australensis, Stylophora pistillata Humicola fuscoatra Acropora formosa, GBR, Caribbean Sea and Indo-Pacific 10,16 Cladocora caespitose Labyrinthula sp. Portites lutea Arabian Sea 9 Mycelial yeast Portites lutea Arabian Sea 9 Paecilomyces godlewski M. cavernosa Barbados (West Indies) 7 Paecilomyces lilacinus G. australensis GBR 7 Penicillium avellaneum Porites porites Barbados (West Indies) 7 Penicillium citrinum Acropora formosa GBR 10 Penicillium expansum Acropora palmata Barbados (West Indies) 7 Penicillium restrictum Porites australensis GBR 7 Penicillium stoloniferum Acropora palmata, Diploria labyrinthiformis Barbados (West Indies) 7 Phialophora bubaki Diploastrea heliopora, Goniastrea retiformis, GBR 7 Porites australensis Phoma sp. Acropora formosa* GBR 10 Pichomyces chartarum Acropora sp. GBR 7 Scolecobasidium sp. Montipora tuberculosa, Porites lutea, Andaman Islands, Bay of Bengal and 14 Goniopora Indian Ocean Thraustochytrium motivum Various coral species Arabian Sea 14 Trichoderma sp. Cladocora caespitose Caribbean Sea and Indo-Pacific 16 Wallemia ichthyophaga Porites sp. GBR 7 5 CORAL ASSOSIATED FUNGI Each polyp is made out of three tissue layers with the mesoglea being flanked by the epidermis and the endodermis17. A circle of tentacles around the mouth help the coral to capture and ingest plankton for food, clear away debris from the mouth, and act as the animals primary means of defence6,17. Stinging cells called nematocysts are located along the surface between the mouth