Fungal diversity of mangrove-associated sponges from New Washington, Aklan, Philippines

ABSTRACT Sponge-associated fungi are the least explored marine fungal groups. It is only in recent years that fungal symbionts of marine sponges have received attention mainly due to the isolation of bioactive metabolites while not much attention was given to their specificity, biogeography and exact roles in marine sponges. The diversity of fungi associated with mangrove sponges (Axinella sp., Halichondria cf. panicea, Haliclona sp., Tedania sp.) collected from New Washington, Aklan, Philippines were investigated using morphological observation. A total of 110 species of sponge-associated fungi belonging to 22 genera of ascomycetes with 18 genera of asexual morphs whose sexual stage is unknown, 2 genera of basidiomycetes, 21 morphospecies of Mycelia sterilia, 1 unidentified yeast species and 11 unidentified hyphomycetes were isolated from four species of mangrove sponges. This is the first study that explored the diversity and ecology of sponge-associated fungi in mangrove habitats from the Philippines. The results of the study suggest host-preference by various fungal taxa and the development of fungi on these hosts appeared to be strongly influenced by the characteristics or nature of the immediate environment.


Introduction
Fungi have long been known to exist in the marine environment but considered to be the underexplored group in the oceans compared to bacteria, plants and animals (Jones and Pang 2012). Marine fungi live as saprophytes, parasites and symbionts on various matrices such as sediments, logs, water, as well as algae, vascular plants, invertebrates and fishes (Johnson and Sparrow 1961;Kohlmeyer and Kohlmeyer 1979). Marine fungi can be classified as true or obligate and facultative marine fungi. The former can complete their lifecycle in marine environments and the latter can grow in freshwater or terrestrial habitats as well as in marine environments (Kohlmeyer and Kohlmeyer 1979). However, Jones et al. (2015) accept a wider interpretation of what can be considered marine.
Sponges are known inhabitants of coastal and deep-sea environments. They are filter feeders and known to harbour diverse groups of microbes (Hentschel et al. 2006;Taylor et al. 2007). Marine sponges are a rich source of compounds with bioactive potential. A review by Blunt et al. (2012) reported that 296 new compounds from marine sponges were isolated only in 2011. But over the past decade, a consensus has developed among experts that these novel natural products from sponge extracts are synthesised, either in part or in entirely, by the symbiotic microbes that are intimately associated with these marine metazoans (Konig et al. 2006;Meenupriya and Thangaraj 2010). These microbes are thought to be involved in a variety of ecological functions including production of secondary metabolites that can contribute to their own ecological success and to that of their host . The previous work on marine microbial diversity in sponges focuses mostly on bacterial associates with proposed novel phylum candidate Poribacteria (Fieseler et al. 2004;Kamke et al. 2014). It is only in recent years that the fungal symbionts of marine sponges have received attention Wang et al. 2008;Baker et al. 2009;Li and Wang 2009;Menezes et al. 2010;Debbab et al. 2011) mainly related to their capacity to produce novel bioactive metabolites (Proksch et al. 2003;Bugni and Ireland 2004;Amagata et al. 2006;Blunt et al. 2009;Aly et al. 2011;Jones 2011).
Fungi have been isolated from subtidal sponges in tropical, subtropical and temperate countries. Wang et al. 2008;Caballero-George et al. 2010;Paz et al. 2010;Zhou et al. 2011;Thirunavukkarasu et al. 2012;Flemer 2013;Henriquez et al. 2013;Bolaños et al. 2015). In Asia, there have been published information on the diversity of sponge-associated fungi in countries like China (Zhang et al. 2009;Liu et al. 2010;Ding et al. 2011;Zhou et al. 2011;Yu et al. 2012;He et al. 2014;Jin et al. 2014), India (Meenupriya and Thangaraj 2010;Thirunavukkarasu et al. 2012) Indonesia (Namikoshi et al. 2002), Israel , Malaysia (Mahyudin 2008) and Russia . To date, there are no ecological studies of sponge-associated fungi or any information about the species richness and fungal biodiversity of sponges in the marine environment of the Philippines. Furthermore, there is no published information about the fungal diversity of mangrove-associated sponges. The present study aims to determine the diversity of fungi associated with different species of mangrove-associated sponges collected from the mangrove area of New Washington, Aklan, Philippines. The assessment of fungal diversity will be of great value to understand the fungal ecology in mangrove-associated sponges and to explore the biotechnological potentials of these fungi. The additional information obtained from this study will serve as baseline information on the fungal composition of the different sponge species in the Philippines.

Study site
New Washington, Aklan is located on the north-eastern part of Panay Island. It is a coastal community composed of islets surrounded by brackish water rivers that forms part of the Batan Estuary and the northern coast of the island faces the Sibuyan Sea. Two sites, Kapispisan (11°38ʹ 5.748" N, 122°25ʹ2.388" E) and Boeo (11°36ʹ 33.804 N, 122°27ʹ 42.119 E), both in Pinamuk-an, New Washington, Aklan, were surveyed for mangrove sponges by mask and snorkel to a depth of 1-2 m in September 2015 ( Figure 1). The mangrove species that dominated Pinamuk-an River include Sonneratia alba, Avicennia umphiana and Avicennia marina (Ochavo et al. unpublished).

Collection and identification of mangroveassociated sponges
Sponges (Axinella sp., Halichondria cf. panicea, Haliclona sp., Tedania sp.) attached to the mangrove roots were collected and photographed for identification purposes. Latex glove was worn for the collection of sponges and samples were removed from the mangrove roots using scalpel or by directly cutting the roots with attached sponge. Sponge samples (~20-25 g) were transferred directly into zip-lock bags containing seawater from the collection sites to prevent direct contact with air. Sponges were stored and transported immediately in a cooler box back to the laboratory for processing within 24 h to avoid external microbial contamination and excessive proliferation of the samples. All the epiphytic faunas attached to the samples were removed.

Isolation of fungi
Sponge-associated fungi were isolated in the Microbiology Laboratory of University of the Philippines Visayas -National Institute of Molecular Biology and Biotechnology (UPV -NIMBB). Sponge tissue segments were rinsed three times with sterile artificial seawater to eliminate adherent surface debris and contaminants . The inner tissue (middle internal mesohyl area) was excised with a sterile scalpel and was cut into small pieces. Ten grams of each sample was homogenised using a blender containing 20 ml sterile artificial seawater under aseptic conditions. One millilitre of the resulting homogenate was transferred to a sterile test tube containing 9 ml of 0.85% NaCl and mixed in the vortex (dilution 10 −1 ). The serial dilution was repeated until 10 −5 has been reached.
For fungal cultivation, 100 µl of dilutions 10 −0 to 10 −5 were plated in triplicate onto 15 ml of the selected isolation media supplemented with penicillin G and streptomycin (100 µg/ml each) using spread plate technique. All five media (Czapek Dox Agar, Cornmeal Agar, Mycobiotic Agar, Potato Dextrose Agar, Rose Bengal Agar) were added with 1.5% NaCl which is the salt concentration used in isolation of mangrove fungi in the Philippines. A control plate was prepared by exposing a blank plate in the middle of the working area for 15 min. The inoculated plates were sealed with Parafilm TM and incubated at room temperature (27°C) in inverted position and examined daily for the appearance of fungal colonies up to three weeks, depending on the growth of species. Edges from emerging fungal colonies growing out on culture medium were picked and transferred with a sterile inoculating loop onto culture tube containing fresh media supplemented with antibiotic solution. Mycelia or spores were transferred again in new culture media for purification. For yeasts, cells were streaked onto a fresh culture media. The resulting plates were incubated at room temperature (27°C) for pure culture and purification was done rigorously until a homogenous fungal isolate was obtained for identification.

Fungal density
The number of colony forming units (CFU) per gram of dry weight of sponge (CFU/g dw) was estimated for each identified fungal species.

Identification of fungal isolates
Filamentous fungi were identified based on their macroscopic (colonial) and microscopic characteristics. Colonial descriptions included colony characteristics such as colour (reverse and obverse), texture, margin, elevation and characteristics of aerial hyphae. Slide culture technique of Riddell (1950) was employed for microscopic examination of fungal isolates that included spore morphology, colour, shape, wall ornamentation or texture, size, conidial formation and other relevant characteristics such as phialide and conidiation pattern. In the Mycology Laboratory of UPV-Freshwater Aquaculture Station, microscopic examination of colony colours and growth rates was assessed with a dissecting microscope. Microscopic characteristics of fungal isolates were determined by viewing slides with distilled water using a light microscope under Low Power Objective (LPO) and High Power Objective (HPO). Microphotographs of the reproductive structures were taken for identification purposes. Filamentous fungi were identified to at least genus level based on the identification scheme by Kohlmeyer (1984), Kohlmeyer and Volkmann-Kohlmeyer (1991), Barnett and Hunter 1998), Howard (2003), Watanabe (2010), Domsch et al. (2007), Pitt and Hocking (2009) and Campbell et al. (2013) in addition to other available keys and monographs. Identification of yeast isolates was based on keys by Kurtzman and Fell (1998) that included colonial and microscopic characteristics and by using API 20C Aux (bioMerieux, Rome, Italy) that was based on 19 carbohydrate assimilation tests with negative control. Mycelia sterilia or fungi that failed to grow and sporulate were given codes using cultural characteristics (e.g. colony surface, texture and hyphal pigmentation). The fungal descriptions in MycoBank (www.mycobank.com) were used as guide for further identification of the fungal isolates.

Preservation of fungal cultures
Continuous growth method was used for fungal culture preservation. After identification, all pure cultures of fungal isolates were grown on agar slant in a culture tube and stored at 5°C. Based on the frequency of occurrence, the following groupings were made (Hyde 1989;Hyde and Sarma 2000;Sarma and Raghukumar 2013): very frequent (≥10%), frequent (5-10%), infrequent (1-5%), rare (≤1%).
(B) The diversity of fungi associated with the mangrove sponges was calculated following Ludwig and Reynolds (1988). (a) Shannon Index H 0 ð Þ ¼ À P ðpi ln piÞ where: p i is the proportion of individuals that species i contribute to the total number of individuals as shown in the formula below: where: P i = proportion of individuals in the ith species As D increases, diversity decreases. Simpson's index is usually expressed as: where: H' max = maximum value of diversity for the number of species present W3here: D = Simpson' index of diversity S = Species richness (A) Jaccard Index of Species Similarity was calculated pair-wise among the hosts based on the presence or absence of each fungal species using the formula where: a is the number of fungal species occurring in both hosts b is the number of fungal species unique to the first host and c is the number of fungal species unique to the second host.
The mangrove sponge Halichondria cf. panicea harboured the most fungal isolates with 54 species, followed by Axinella sp. (45 isolates The Jaccard's coefficient of similarity (J'), based on the presence or absence of each fungus, was calculated among different sponge species to compare the composition of fungi on each sponge host (Table 2). Based on pair-wise comparison of similarities of 110 fungal isolates on four sponges, the similarity index was highest between Halichondria cf. panicea vs Axinella sp. (0.22) followed by both Halichondria cf. panicea vs Tedania sp. and Halichondria cf. panicea vs Haliclona sp. with the J' value of 0.19. It was least between Axinella sp. and Haliclona sp. with J' value of 0.13.

Association of fungi on the different mangroveattached sponges
Three classifications were done based on the presence of fungal genera in certain number of sponge species, as adapted from Li and Wang (2009). "Spongegeneralists" are genera that can be found in all sponge species and results showed that, the genera Acrodontium, Aspergillus, Candida, Paecilomyces and  Penicillium could be classified under this group. The genera Acremonium, Cladosporium, Hortaea and Trichoderma are classified as sponge-associates since they were identified on more than one sponge. The "sponge-specialists" would include the genera Beauveria, Cryptococcus, EuPenicillium, Geotrichum, Gliomastix, Kloeckera, Mammaria, Neosartorya, Pestalotiopsis, Pichia, Ramichloridium, Rhinocladiella, Scedosporium, Stachybotrys and Tritirachium.

Fungal load of mangrove-associated sponges
The total fungal load of the four species of mangrove-associated sponges yielded varying counts on the various culture media used (Table 3). For Halichondria cf. panicea, the highest fungal density was recorded from cornmeal agar and lowest value from potato dextrose agar. The highest CFU in Axinella sp. was recorded from CMA and lowest fungal load from MBA. MBA had the highest fungal load in Tedania sp. and lowest value from CMA. For Haliclona sp., RBACl had the highest fungal load value and lowest CFU from MBA.

Fungal diversity in mangrove-associated sponges
The study on the ecological role, including its diversity and association, of fungi on marine sponges are still scarce and data were largely generated due to the diversity of novel bioactive metabolites produced with promising biotechnological applications Bugni and Ireland 2004;Konig et al. 2006 Table 3. Fungal density (CFU g-1) of different mangrove-associated sponges in five culture media.
These mangrove sponges were collected in the same location but the composition of fungal genera differs from one another except Acrodontium, Aspergillus, Candida, Paecilomyces and Penicillium that were isolated in all sponge species. The genera Beauveria, Cryptococcus, Eupenicillium, Kloeckera, Pichia, Stachybotrys were only isolated in Tedania sp.; Geotrichum and Gliomastix in Axinella sp.; Mammaria, Neosartorya, Tritirachium in Haliclona sp.; Pestalotiopsis, Ramichloridium, Rhinocladiella, Scedosporium in Halichondria cf. panicea. The differences in the fungal composition on the different sponge species suggest host-preference of the different fungal taxa. Furthermore, the results in this study and previous works in subtidal sponges Wang et al. 2008;Liu et al. 2010;Yu et al. 2012) showed that the differences in the fungal composition and its diversity may be attributed to the species of sponges with various morphological structures. Ding et al. (2011), on his work on South China Sea sponges (Clathrina luteoculcitella and Holoxea sp.) sampled in the same location, also observed this wherein orders Agaricales, Boliniales, Microascales, Mucorales, Pleosporales, Saccharomycetales and Xylariales were only isolated from sponge Clathrina luteoculcitella but not in Holoxea sp. Thus, even the sponges were collected on the same location, they harbour different isolates which suggested that these isolates were not spores from seawater column and trapped during the filter feeding process of sponges. Previous studies by Gao et al. (2008), Wang (2009) andJin et al. (2014) demonstrated that fungal communities isolated from sponges differ from the surrounding water. For example, Penicillium janthinellum, Fusarium solani and P. chrysogenum which were isolated from seawater samples but not present within sponges ). However, it is insufficient to disprove sponge-specific nature of a microbe by merely proving the presence of microbe outside a sponge. A predator or storm for example may damage sponge and microbes associated with it may disintegrate and spread into the seawater column (Taylor et al. 2007).
Diversity of fungi associated with marine sponges remains an understudied area and more evidence is required to elucidate their possible ecological role Wang et al. 2008). The present investigation does not show any direct evidence that the isolated fungi have been actively growing on the sponge tissues. As a result, the difficulty also arises on how to determine whether they are sponge-symbiotic fungi or not. So far, there is little evidence regarding the symbiotic relationship between sponges and fungi. For example, Maldonado et al. (2005) showed direct evidence of sponge-endosymbiotic yeasts in a marine sponge Chondrilla sp. that is transmitted maternally through fertilised eggs based using immunocytochemical technique to label the β-1,4-N-acetyl-D-glucosamine residues of chitin walls. There is also indirect evidence of the putative fungal original intron in a sponge Tetilla sp., as observed by Rot et al. (2006), perhaps because of horizontal gene transfer. In addition, marine ascomycetes of the genus Koralionastes have been reported to be in some way associated with crustaceous sponges wherein it forms fruiting bodies only in close association with the sponges associated with corals (Kohlmeyer and Volkmann-Kohlmeyer 1990). Furthermore, Perovic-Ottstadt et al. (2004) demonstrated the presence of receptor proteins for fungal cell wall components (e.g. (1→3)β-d-glucan-binding proteins), in the marine sponge Suberetis domuncula, which indicates that sponges are biochemically equipped for dealing with fungi. Several bacteria and archaea, along with the ubiquitous fungus Penicillium, as reported by Simister et al. (2012), are symbionts of sponges. Using immunocytochemistry, transmission electron microscopy (TEM) technique and non-cultivation-dependent analysis, the real association between fungi and marine sponges will be confirmed including vital role or functions of fungi in the sponge (Maldonado et al. 2005;Passarini et al. 2013). Höller et al. (2000) proposed that investigation of a larger number of samples and surrounding water should be done to determine if sponge-associated fungi are not terrestrial fungi filtered from the surrounding waters but adapted to the marine habitat and on its host.
Even employing diverse culture media, 21 species remained sterile. Höller et al. (2000) also isolated 37 strains of Mycelia sterilia in 14 sponges even using diverse culture media and culture conditions to induce sporulation of fungi. Molecular analysis can be of great help to identify fungi with no reproductive structures (e.g. conidia and ascomata) Furthermore, 11 species of hyphomycetes remained unidentified and requires further investigation including molecular analysis for identification.
It is difficult to suggest that the present study isolated sponge-specialist based on the differences of fungi recovered from four mangrove sponges because there is no direct evidence and there are limited studies on fungal associates of mangrove sponges. Extensive survey of fungi in more species of sponges including comparison on the same species in this study but different geographical locations and using biochemical and molecular methods (e.g. 454 pyrosequencing) could reveal the sponge-fungal association.
In addition, the isolated fungal genera are common to terrestrial habitats, suggesting that these isolates may also be of terrestrial origin and can be considered, based on the definition of Kohlmeyer and Kohlmeyer (1979), facultative marine fungi but on the latest definition of Pang et al. (2016), these isolates were considered marine fungi. Marine fungi, as defined by Pang et al. (2016), are fungi that are recovered repeatedly from marine habitats because: (1) it can grow and/or sporulate (on substrata) in marine environments; (2) it forms symbiotic relationships with other marine organisms or (3) it is shown to adapt and evolve at the genetic level or be metabolically active in marine environments. If we based on the list of marine fungi by Jones et al. (2015), 16 species were considered marine fungi that include 9 species of Aspergillus (A. candidus, A. niger, A. ochraceus, A. cf. penicilloides, A. restrictus, A. sclerotiorum, A. sydowii, A. tamarii, A. terreus), 3 species of Penicillium (P. cf. citreonigrum, P. cf. citrinum, P. spinulosum), 1 Trichoderma species (T. aureoviride), 1 species of Candida (C. guilliermondii) and 2 species of Cladosporium (C. cladosporioides, C. sphaerospermum).

Environment-dependent fungal diversity
The results of the present work and earlier studies show that the diversity sponge-associated fungi are more dependent on the surrounding environment where the sponge species thrives. For instance, no ascomycetes were isolated in mangrove sponge Halichondria cf. panicea collected from Aklan, Philippines while previous works of Höller et al. (2000) in Helgoland, Germany and Pivkin et al. (2006) in Sakhalin Island, Russia recovered ascomycetes in the subtidal sponge Halichondria panicea. Both present work and other published studies on Halichondria harbours Acremonium, Aspergillus, Penicillium, Mycelia sterilia and Trichoderma. Only Höller et al. (2000) isolated Mucor, a zygomycete. Furthermore, Flemer (2013) and Bolaños et al. (2015) recovered ascomycetes in subtidal sponges under the genus Axinella while no ascomycetes were isolated from mangrove sponge Axinella sp. collected in Aklan, Philippines. Only Cladosporium was isolated in the present work and two former studies in Axinella dissimilis (Flemer 2013) and Axinella sp. 1 . There is no similar species from the present work were recovered in Axinella sp. 2 and Axinella sp. 3 . Furthermore, between the sponge Haliclona simulans, collected from the coastal waters of Ireland ), and Hainan Province of China . The fungal diversity between two different regions is quite different. For instance, the orders Capnodiales, Dothideales, Agaricostilbales, Wallemiales, which were present in the "Hainan" sample are not found in the "Irish" sample. In the "Irish" sample, fungi under the orders Chaetosphaeriales, Chaetothyrailes, Helotiales, Mucorales and Agaricomycotina, were isolated but were absent in the "Hainan" sample. Furthermore, no shared identical fungal species were observed in the two collections. The results of the comparison of the abovementioned studies supports the notion that being filter feeders, sponges enrich various fungal species from the surrounding seawater that are merely washed into the sea from their terrestrial habitats and just happen to survive in their "host organisms" Taylor et al. 2007;Proksch et al. 2008;Wang et al. 2008;Liu et al. 2010;Wiese et al. 2011;Zhou et al. 2011). These remain dormant until plated onto a suitable culture medium ). If such has been the case, the metabolic activities of fungi from sponges should be the same as that of those in other terrestrial environments. Surprisingly, these facultative marine fungi produce novel compounds that are different and not produced from their terrestrial conspecifics (Proksch et al. 2003;Konig et al. 2006;. Secondary metabolites in producing fungi play an important role in ecological interactions with other organisms allowing it to survive in its ecological niche while on its host, it enhance the defence mechanisms against pathogens and predators (Fox and Howlett 2008;Thomas et al. 2010).

Conclusion
The fungal composition differs in each species of mangrove sponges even though they were collected in the same location suggesting that the isolates recovered were not merely seawater contamination and suggest sponge-preference by various fungal taxa that can be classified as true marine fungi. The development of marine fungi on these hosts appeared to be strongly influenced by the characteristics or nature of immediate environment.