Biodiversity and Enzyme Activity of Marine Fungi with 28 New Records from the Tropical Coastal Ecosystems in Vietnam

Figures & data

Figure 1. Sampling positions at Nha Trang Bay and Van Phong Bay of Khanh Hoa Province, Vietnam. Detail information on locations was given in the Table 1.

Table 1. Details regarding colony forming units (CFUs) of fungal colonies on the SDA medium plates and strains identified with ITS sequence.

Sampling date	Location	Lat (°N)	Long (°E)	Habitat	Depth (m)	pH	Temperature (°C)	Salinity (ppt)	CFU L^–1	No. of colonies (strains)
										Yeast	Filamentous fungi	Total
07/05/2017	Nha Trang Central Beach (NB)	12^o14′	109^o11′	Coastal marine waters (CMW)	0.03–0.05	7.95	29	1.6	6.0 × 10^2	6 (5)	2 (2)	8 (7)
07/05/2017	Song Cai River Estuarine (SC)	12^o15′	109^o11′	CMW	0.03–0.05	8.15	28	3.5	2.0 × 10^3	6 (2)	7 (5)	13 (7)
20/5/2017	Doc Let Beach (DL)	12^o33′	109^o13′	CMW	0.03–0.05	7.79	31	3.45	9.3 × 10^2	10 (5)	2 (2)	12 (7)
24/5/2017	Dam Mon Lagoon (DM)	12^o40′	109^o24′	CMW	0.03–0.05	8.4	32	3.5	2.4 × 10^4	10 (7)	3 (3)	13 (10)
24/5/2017	Tu Bong River Estuarine (TB)	12^o46′	109^o19′	CMW	0.03–0.05	8.38	33.5	3.5	3.1 × 10^4	7 (5)	4 (4)	11 (9)
24/5/2017	Ran Trao Marine Ecosystem Reserve (RT)	12^o37′	109^o12′	CMW	0.03–0.05	7.96	32	3.25	1.4 × 10^4	16 (3)	1 (1)	17 (4)
09/06/2017	Cau Da Port (CD)	12^o12′	109^o13′	CMW	0.03–0.05	8.01	29.5	3.3	5.4 × 10^4	19 (4)	3 (2)	22 (6)
10/8/2017	Vung Ngan Aquaculture Zone (VN)	12°11′06"	109°16′25"	CMW	0.03–0.05	8.01	29	3.25	5.7 × 10^3	13 (2)	5 (4)	18 (6)
10/8/2017	Hon Mun Core Zone (HMC)	12°10′33"	109°18′16"	CMW	0.03–0.05	7.97	29	3.3	1.1 × 10^5	21 (3)	1 (1)	22 (4)
10/8/2017	Hon Mun Buffer Zone (HMB)	12°10′72"	109̊18′56”	CMW	0.03–0.05	7.96	30	3.4	1.8 × 10^5	7 (2)	4 (4)	11 (6)
01/6/2017	Deep Waters (DW)	12°18′31′’	109°31′67′’	Deep waters	40–47	7.0	22	3.44	8.8 × 10^2	0 (0)	16 (9)	16 (9)
Total										115 (38)	48 (37)	163 (75)

Table 2. Taxonomy of cultured fungal species found in this study.

No.	Division	Subdivision	Class	Order	Family	Genus	Species	Strain^1	Relative abundance (%)
1	Ascomycota	Pezizomycotina	Dothideomycetes	Capnodiales	Cladosporiaceae	Cladosporium	C. colombiae-like	DM11M1, NB8M	2.67
2							C. cycadicola	DW8M	1.33
3							C. sphaerospermum	VN16M	1.33
4				Dothideales	Saccotheciaceae	Aureobasidium	A. melanogenum	DL9Y	1.33
5				Pleosporales	Didymellaceae	Stagonosporopsis	S. oculi-hominis-like	DW3M	1.33
6					Pleosporaceae	Alternaria	A. alstroemeriae	CD22M	1.33
7			Eurotiomycetes	Chaetothyriales	Cyphellophoraceae	Cyphellophora	C. oxyspora	HMB10M	1.33
8				Eurotiales	Aspergillaceae	Aspergillus	A. aflatoxiformans/ austwickii-like	TB8M, TB9M DW1M, DW2M	5.33
9							A. costaricaensis-like	TB10M	1.33
10							A. uvarum/ japonicus-like	DM11M2, DM12M	2.67
11							A. alabamensis	HMB8M, HMB9M VN15M	4.00
12							A. versicolor-like	VN17M, VN18M, HMB11M	4.00
13						Penicillium	P. paxilli	NB7M	1.33
14							P. steckii	SC7M	1.33
15							P. citrinum	CD20M	1.33
16							P. oxalicum	TB11M	1.33
17							P. simplicissimum-like	DL11M	1.33
18							P. cuddlyae-like	DW14M	1.33
19			Sordariomycetes	Hypocreales	Cordycipitaceae	Parengyodontium	P. album	DW11M DW13M	2.67
20						Simplicillium	S. obclavatum	DW15M	1.33
21					Hypocreaceae	Trichoderma	T. lixii	SC9M	1.33
22							T. yunnanense	SC11M	1.33
23							T. atrobrunneum/ lixii-like	SC13M	1.33
24					Nectriaceae	Fusicolla	F. acetilerea-like	DW7M	1.33
25				Trichosphaeriales	Trichosphaeriaceae	Nigrospora	N. sphaerica	HMC22M	1.33
26				Sordariales	Sordariaceae	Gelasinospora	G. saitoi	RT3M	1.33
27		Saccharomycotina	Saccharomycetes	Saccharomycetales	Debaryomycetaceae	Candida	C. blankii	15^2	20.00
28							C. tropicalis	6^3	8.00
29							C. akabanensis	TB1Y, TB6Y	2.67
30							C. intermedia	DL6Y DL8Y	2.67
31						Millerozyma	M. farinosa	NB1Y	1.33
32						Meyerozyma	M. caribbica	TB3Y	1.33
33						Limtongozyma	L. cylindracea	DL7Y	1.33
34					Pichiaceae	Pichia	P. kudriavzevii	TB5Y, DM4Y, VN9Y	4.00
35					Metschnikowiaceae	Kodamaea	K. ohmeri	DM2Y DM9Y	2.67
36	Basidiomycota	Agaricomycotina	Agaricomycetes	Polyporales	Phanerochaetaceae	Phanerochaete	P. chrysosporium-like	DL12M	1.33
37		Pucciniomycotina	Microbotryomycetes	Sporidiobolales	Sporidiobolaceae	Rhodotorula	R. mucilaginosa	NB2Y, CD18Y	2.67
38							R. diobovata	DM5Y	1.33
39						Rhodosporidiobolus	R. fluvialis	CD19Y	1.33
40	Mucoromycota	Mucoromycotina	Mucoromycetes	Mucorales	Mucoraceae	Hyphomucor	H. assamensis	SC10M	1.33
Total	3	5	7	12	17	22	≥ 40	75	100

¹Strains identified by molecular data in this study. ²15 Candida blankii strains: SC2Y, CD1Y, CD2Y, DM6Y, DM7Y, DM8Y, RT5Y, RT6Y, RT7Y, HMC1Y, HMC2Y, HMC11Y, HMB1Y, HMB7Y, VN1Y; and ³6 Candida tropicalis strains: NB3Y, NB5Y, NB6Y, SC1Y, DL5Y, TB4Y.

Table 3. Phylogenetic affiliation and taxonomy of the fungal OTUs obtained with ITS primers based on cultivated-independence studies.

GenBank Accession	Final taxon	Phylum	Function	Closest relative	GenBank Accession	Identity (Coverage) %	Originally reported habitat	Abundance count				Abundance (%)
								CD	HMC	DW	Average	CD	HMC	DW	All
MT364509	Candida_blankii OTU_2	Ascomycota	Pathotroph: Human, animal and plant Pathogen	C.blankii isolate IIC1M.1	MF940143	100 (100)	Sands of Copacabana’s beach, Rio de Janeiro, Brazil	2467	51,651	0	18039.33	95.95	97.99	0	97.74
				C. blankii CD1Y, CD2Y, HMC1Y, HMC2Y and HMC11Y	MT102799 MT102800 MT102807 MT102808 MT102809	99.65–100 (95)	CD and HMC samples in this study (direct matching)
MT364534	Aspergillus_versicolor OTU_29	Ascomycota	Saprotroph: Aliphatic and aromatic hydrocarbon degradation	Aspergillus versicolor HT-M36	KJ527011	100 (100)	Fen-Daqu fermentation starter (China)	43	782	0	275.00	1.67	1.48	0	1.49
MT364570	Epicoccum_sorghinum OTU_65	Ascomycota	Possible plant pathogen	Epicoccum sorghinum HM19B	MT125854	100 (100)	Dark Leaf Spot Diseased Rice	0	250	0	83.33	0.00	0.47	0	0.45
MT364606	Chytridiomycota OTU_101	Chytridiomycota	Unknown	Uncultured fungus clone S12T_70	KU163812	100 (94)	Sea water in China	0	0	87	29.00	0.00	0.00	100	0.16
MT364609	Fungal OTU_104	Unclassified Fungi	Possible phytoplankton pathogen	Uncultured fungus isolate DGGE gel band F-SW39	KC491368	90.3 (100)	Sea water in South Florida, Crab Cove, USA	61	27	0	29.33	2.37	0.05	0	0.16
Total								2571	52,710	87	18,456	100	100	100	100

Figure 2. The percentage abundance of major phyla of fungi isolates from all 11 samples using culture-dependent approach (A), fungi isolates from 3 samples HMC, CD and DW using culture-dependent approach (B), and fungal OTUs from 3 samples HMC, CD and DW using culture-independent approach (C).

Figure 3. Phylogenetic tree of Orders Capnodiales, Dothideales and Pleosporales (class Dothideomycetes, Phylum Ascomycota) based on ITS rRNA genes. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model [54]. The tree with the highest log likelihood –2876.51) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 34.52% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 18 nucleotide sequences, including ones in this study (*), from type strains (T) and Chytridium olla UACCC ARG100 (T) used as an outgroup.

Figure 4. Phylogenetic tree of Orders Chaetothyriales and Eurotiales (Class Eurotiomycetes, Phylum Ascomycota) based on ITS rRNA genes. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model [55]. The tree with the highest log likelihood (–3375.53) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3870)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 41 nucleotide sequences, including ones in this study (*), from type strains (T) and Chytridium olla UACCC ARG100 (T) used as an outgroup.

Figure 5. Phylogenetic tree of Orders Hypocreales, Trichosphaeriales and Sordariales (Class Sordariomycetes, Phylum Ascomycota) based on ITS rRNA genes. The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model [54]. The tree with the highest log likelihood (–3297.12) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.3655)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 18 nucleotide sequences, including ones in this study (*), from type strains (T) and Chytridium olla UACCC ARG100 (T) used as an outgroup.

Figure 6. Phylogenetic tree of Order Saccharomycetales (Class Sacharomycetes, Phylum Ascomycota) based on ITS rRNA genes. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura 3-parameter model [54]. The tree with the highest log likelihood (–3834.69) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.5029)). This analysis involved 42 nucleotide sequences, including ones in this study (*), from type strains (T) and Chytridium olla UACCC ARG100 (T) used as an outgroup.

Figure 7. Phylogenetic tree of Order Polyporales (Class Agaricomycetes, Phylum Basidiomycota), Order Sporidiobolales (Class Microbotryomycetes, Phylum Basidiomycota) and Order Mucorales (Class Mucoromycetes, Phylum Mucoromycota) based on ITS rRNA genes. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura 3-parameter model [54]. The tree with the highest log likelihood (–3580.61) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura 3 parameter model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.8620)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 14 nucleotide sequences, including ones in this study (*), from related type strains (T) and Chytridium olla UACCC ARG100 (T) used as an outgroup.

Figure 8. Relative extracellular enzyme activity (AU) of all fungal strains from each sample at Nha Trang Bay and Van Phong Bay, Khanh Hoa province, Vietnam. For each strain, 1 AU was defined as an enzyme activity unit with hydrolysis zone diameter D–d = 10–15 mm, 2 AU for D–d = 15-20 mm and 3 AU for D–d > 20 mm. Based on total relative enzyme activity shown, the samples were divided into 5 groups: Group A (>60 AU), Group B (23–33 AU), Group C (20 AU), Group D (12–16 AU), and Group E (0–5 AU).

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