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Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing
Deep-Sea Research I 116 (2016) 99–105
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Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing Xiao-Yong Zhang a,b,c, Guang-Hua Wang a,b,c, Xin-Ya Xu a,b,c, Xu-Hua Nong a,b,c, Jie Wang a,b,c, Muhammad Amin a,b,c, Shu-Hua Qi a,b,c,n a Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China b Guangdong Key Laboratory of Marine Material Medical, South China Sea Institute of Oceanology, Chinese Aacademy of Sciences, 164 West Xingang Road, Guangzhou 510301, China c RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 May 2016 Received in revised form 6 August 2016 Accepted 16 August 2016 Available online 17 August 2016
The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deepsea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Fungal diversity Deep-sea sediments Okinawa Trough Illumina sequencing ITS1
1. Introduction Although there is only about 1% of the photosynthetically produced organic carbon that reaches to the deep-sea floor, these deep-sea sediments are now recognized as a home to rich and diverse microbial communities which provide a fundamental contribution to global carbon recycling through the sequestration and remineralization of organic matter (Jamieson et al., 2013). The presence and ecological importance of bacteria in deep-sea sediments have been well recognized ever since Zobell and Morita (1957) isolated bacteria specifically adapted to grow under high pressures and termed them “piezophile” (Whitman et al., 1998; Li et al., 1999; Sogin et al., 2006). However, the fungi in deep-sea n Corresponding author at: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China. E-mail address: [email protected] (S.-H. Qi).
http://dx.doi.org/10.1016/j.dsr.2016.08.004 0967-0637/& 2016 Elsevier Ltd. All rights reserved.
sediments have been neglected previously. One of the first reports of fungi in deep-sea sediments was provided by Kohlmeyer and Kohlmeyer (1979), who reported obligate marine fungi from wooden panels immersed at depths of 500–3000 m. Since then, fungi have been found in different deep-sea sediments from the Mariana Trench (Takami et al., 1997; Nagano et al., 2010), Chagos Trench (Raghukumar et al., 2004), Indian Ocean (Damare et al., 2006; Singh et al., 2011 and 2012; Zhang et al., 2014), South China Sea (Lai et al., 2007; Zhang et al., 2013), Peru Trench (Edgcomb et al., 2011), Gulf of Mexico (Thaler et al., 2012) and Pacific Ocean (Xu et al., 2014 and 2016). Despite of the recent advances, the knowledge and understanding pertaining to the diversity and distribution of fungal communities in deep-sea sediments is still limited. The early studies on fungal diversity in deep-sea sediments were mainly based on cultivation techniques (Damare et al., 2006; Le Calvez et al., 2009; Jebaraj et al., 2010). Fungal species detected in deep-sea sediments by culture-dependent methods mostly
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belonged to the phylum Ascomycota and limited yeast species of Basidiomycota (Nagano and Nagahama, 2012). However, the traditional molecular approach (clone library analysis) indicated that cultivable fungi were only a small fraction of the total fungi in deep-sea sediments (Nagano et al., 2010). Recently, an increasing number of novel fungal phylotypes including DSF-group and BCGI clade (Damare et al., 2006), KML11 clade and Rozella (Burgaud et al., 2009) have been detected by clone library analysis. However, this technique of clone library analysis represents a low throughput, costly and tedious method, providing only limited information of the diversity associated with the number of clones analyzed (Monchy et al., 2011). With the recent development of DNA sequencing technologies, high-throughpu sequencing of rRNA gene fragments has been introduced to the field of fungal ecology, which can detect much deeper fungal community in diverse environments, such as soil (Buee et al., 2009) and phyllosphere (Jumpponen and Jones, 2009; Jumpponen et al., 2010). In this study, we presented the use of high-throughpu Illumina sequencing to assess the fungal diversity in deep-sea sediments from Okinawa Trough.
2. Materials and methods 2.1. Study site and sampling Four deep-sea sediment samples (A–D) were collected using electro hydraulic grab with underwater television camera (for samples A and C) and box sampler (for samples B and D) during the Okinawa Trough Cruise. Latitudes and longitudes, and depths of the collected deep-sea sediment samples A–D were 27°48.47'N and 126°54.32'E, 1190 m; 27°48.12'N and 126°58.89'E, 1330 m; 27°33.07'N and 126°58.36'E, 1387 m; 27°34.01'N and 126°55.59'E, 1589 m; respectively (Zhang et al., 2015a). The collected sediment samples were mostly undisturbed and compact. The average length of the sediment cores collected from these locations was approximately 30 cm. Subsections of 3 cm down to 8 cm were cut from the sediment sub-cores and immediately stored in sterile plastic bags to avoid any aerial contamination. The bags were sealed and stored at –20 °C until processing of direct DNA extraction (Zhang et al., 2014). The distances of samples A and B from the active hydrothermal vents in Iheya North were about 1.1 km and 6.3 km, respectively. While the distances of samples C and D from the active hydrothermal vents in Iheya Ridge were about 0.4 km and 4.7 km, respectively. The geochemical properties (including total organic carbon (TOC), total nitrogen (TN), total sulfur (TS), and several metals) of the four sediment samples were described by Zhang et al. (2015a). 2.2. DNA extraction and Illumina sequencing Total genomic DNA from two grams of sediment samples was extracted directly using the FastDNAs spin kit (MP bio, Santa Ana, USA) according to the manufacturer's instruction. DNA concentration was then determined using a NanoDrop ND-2000 UV– vis spectrophotometer (Thermo Scientific, Wilmington, USA). According to the concentration, DNA was diluted to 1 ng/μL using sterile water. Total genomic DNA was amplified using the primers ITS1F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2 (5′GCTGCGTTCTTCATCGATGC-3′) that amplified the internal transcribed spacer-1 (ITS1) region of the fungal rRNA gene (White et al., 1990). PCR reactions were carried out in 30 μL reactions with 15 μL of Phusions High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of forward and reverse primers, and about 10 ng templates DNA. Thermal cycling consisted of initial denaturation
at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and elongation at 72 °C for 2 min, and a final extension at 72 °C for 10 min. The presence of PCR products was determined by analyzing 3 μL of product on 2% agarose gel. Samples with bright main strip between 100 and 500 bp were chosen for further experiments. PCR products were mixed in equidensity ratios. Then, mixture of PCR products were purified with GeneJET Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using NEB Nexts Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the [email protected] Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina MiSeq platform (Version 3) at Novogene Bioinformatic Technology Co., Ltd, Beijing, China. 2.3. OTU cluster and species annotation The raw Illumina sequences were assigned to individual samples based on their unique barcodes. The barcodes and ITS1 primer sequence were then removed to generate Paired-end reads (about 250 bp). Raw tags were then generated by merging Paired-end reads with FLASH, a very fast and accurate analysis tool (Magoc and Salzberg, 2011). The raw tags were quality filtered and analyzed with the Quantitative Insight Into Microbial Ecology (QIIME) software package (Caporaso et al., 2010). Operational taxonomic units (OTUs) were assigned using QIIME's Uparse v7.0.1001 with a threshold of 97% pairwise identity (Edgar, 2013). OTUs were classified taxonomically using QIIME-based wrapper of the Ribosomal Database Project (RDP) classifier (Wang et al., 2007) against the UNITE database (http://unite.ut.ee/, Version 2014.05.13). Any OTUs representing non-fungal sequences were removed before downstream analysis (Bokulich et al., 2013). 2.4. Diversity analysis OTUs abundance information was normalized using a standard of sequence number corresponding to the sample with the least sequences (Gihring et al., 2011). Subsequent analysis of alpha diversity (with-in sample species richness) and beta diversity (between-sample community dissimilarity) was performed basing on this output normalized data. In order to reveal alpha diversity, we rarified the OTU table and calculate three metrics: Chao1 estimates the species abundance; Observed Species estimates the amount of unique OTUs found in each sample, and Shannon's index provides a means of comparing the diversity between two or more samples (Schloss et al., 2009). Rarefaction curves were generated based on these three metrics. In beta diversity analysis, cluster analysis was preceded by principal component analysis (PCA) (Jolliffe, 1986) and Bray-Curtis dissimilarity (Bray and Curtis, 1957) was calculated using the QIIME software package. 2.5. Nucleotide sequence accession numbers Raw sequences of samples A–D were deposited in the Sequence Read Archives of the NCBI under accession number SRR 3234033– 3234036.
3. Results 3.1. Illumina sequencing and sequence analysis After denoising and chimera detection, a total of 41,801 sequences were obtained. After removed 1504 non-fungal sequences
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diversity indices (H′) were also calculated, ranging from 1.2 to 6.1. 3.2. Fungal community composition
Fig. 1. Rarefaction curves constructed for fungal ITS1 (internal transcribed spacer 1) sequences recovered from four deep-sea sediment samples (A–D) from Okinawa Trough by high-throughpu Illumina sequencing. Table 1 Comparison of the estimated operational taxonomic unit (OTU) richness, diversity indexes of the fungal ITS1 sequences for clustering at 97% sequences similarity from the sequencing analysis. Sample Name
Observed OTU
Chao1
Shannon
A B C D
37 112 139 241
44.273 184.565 158.029 310.400
1.613 2.819 4.050 6.122
(consisting of plant-associated sequences), a total of 40,297 fungal ITS1 sequences were retained and used for analysis, which were clustered into 420 OTUs with 97% sequence similarity. The numbers of fungal ITS1 sequences and OTUs detected in samples A–D were 3324 and 37, 17872 and 112, 3352 and 139, and 15749 and 241, respectively. Rarefaction curves were constructed for the number of ITS1 sequences vs OTUs (Fig. 1). Only sample A demonstrated a plateau, indicating that the number of sequences analyzed might sufficiently represent the fungal diversity in the sample. This result was confirmed by a comparison of the observed and estimated OTUs (Chao 1 indices) at the 97% sequence similarity level. With the Chao 1 indices, the numbers of observed OTUs were close to those estimated for samples A and C. While for the other two samples (B and D), the numbers of observed OTUs were lower than the estimated OTUs (Table 1). The Shanon's
A total of 170 fungal taxa detected in four deep-sea sediment samples covered a wide variety of organisms from four formally described fungal phyla and several unassigned fungal phyla. About 78% of the total fungal sequences belonged to Ascomycota, followed by Basidiomycota (17.3%), and a small proportion belonged to Zygomycota (1.5%), Chytridiomycota (0.8%) and several unassigned fungal phyla (2.4%) (Fig. 2). These taxa were mainly distributed in eight fungal classes: Sordariomycetes (46%), Dothideomycetes (8%), Eurotiomycetes (6%), Leotiomycetes (3%), Pezizomycetes (2%), Agaricomycetes (7%), Tremellomycetes (2%) and Chytridiomycetes (0.7%) (Table 2). Among the 50 most abundant fungal taxa (Table 2), 17 taxa (31.7%) could be identified as unassigned fungal genera in the UNITE database (http://unite.ut.ee/, Version 2014.05.13); and 21 taxa (20.2%) were common fungal genera frequently found in different deep-sea environments (Nagano et al., 2010; Damare et al., 2006; Zhang et al., 2014; Jebaraj et al., 2010; Nagahama and Nagano, 2012; Burgaud et al., 2009), while the remaining 12 taxa (21.5%) were rarely recovered in deep-sea environments (Table 2). 3.3. Comparison of the fungal community structures in different samples The Bray-Curtis analysis showed 83–94% dissimilarity of fungal communities between each two sediment samples (Fig. 3). There were 14, 66, 32 and 132 unique OTUs in samples A, B, C and D, respectively. Only 3 OTUs affiliated with fungal genera Pseudogymnoascus, Stemphylium and Chaetomium, were shared among the four samples. The principal component analysis clearly showed variations in fungal community among different sediment samples (Fig. 4). Comparative analysis of the fungal community structures in the four sediment samples revealed a distinct distribution of fungal phyla (Fig. 2). For instance, Ascomycota accounted for 75–95% of the total sequences in samples A, B and D, whereas this phylum accounted for only 49% of the total sequences in sample C (Fig. 2). Alternatively, sample C was characterized by a relatively higher percentage of Basidiomycetous fungi compared with the other samples. In addition, Zygomycota was mainly found in samples C and D, while Chytridiomycota was recovered in samples B and D. To further compare the distinct communities of the four samples, taxonomic composition profile of different four samples were analyzed based upon the 20 most abundant ITS1 sequences in the
Fig. 2. Relative abundance of the fungal phyla recovered from four deep-sea sediment samples (A–D) from Okinawa Trough by high-throughpu Illumina sequencing.
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Table 2 The 50 most abundant fungal taxa (accounted for 73.4%) in the Illumina sequencing library. No. taxon
Phylum
Class
Genus
No. OTU
Similarity %
GenBank accession
Totala
1c 2c 3 4 5 6 7 8 9 10 11c 12 13c 14 15c 16 17c 18c 19 20 21c 22 23 24 25 26 27 28 29 30c 31 32 33 34 35 36 37 38c 39 40 41 42 43 44c 45c 46c 47c 48c 49 50
Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Basidiomycota Basidiomycota Ascomycota Basidiomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota unassigned Zygomycota Ascomycota unassigned Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Basidiomycota Basidiomycota Ascomycota Ascomycota Ascomycota Basidiomycota Zygomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Chytridiomycota
Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Leotiomycetes Sordariomycetes Pezizomycetes unassigned Sordariomycetes Agaricomycetes Tremellomycetes Dothideomycetes Agaricomycetes Eurotiomycetes Sordariomycetes Eurotiomycetes Eurotiomycetes Dothideomycetes Eurotiomycetes Dothideomycetes unassigned incertaesedis Dothideomycetes unassigned Sordariomycetes Dothideomycetes Sordariomycetes Dothideomycetes Sordariomycetes Agaricomycetes Agaricomycetes Dothideomycetes Sordariomycetes Sordariomycetes Agaricomycetes incertaesedis Dothideomycetes Dothideomycetes Eurotiomycetes Eurotiomycetes Dothideomycetes Leotiomycetes incertaesedis Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Chytridiomycetes
unassigned Myriogenosporab Faurelinab Lecythophora Coniellab Thelebolusb Fusarium unassigned unassigned Plectosphaerellab Russulab unassigned Leptosphaeria unassigned Aspergillus Fusarium Exophiala Penicillium unassigned Paecilomyces Preussiab unassigned Mortierella unassigned unassigned unassigned unassigned unassigned Pseudogymnoascusb Myrotheciumb Schizophyllum unassigned Corynespora unassigned Trichocladium unassigned Mortierella Phoma Lophiostoma Emericella unassigned Stemphyliumb unassigned Minimidochiumb Acremonium Nectria Chaetomium Podospora Microdochium Phlyctochytriumb
300 5 33 286 3 10 55 9 11 7 275 106 180 186 170 101 234 81 112 43 23 111 154 31 45 83 37 155 51 314 87 42 66 20 12 378 60 53 82 363 450 80 48 160 70 49 223 100 92 41
97 98 100 100 100 100 100 98 89 100 100 100 100 96 100 100 100 100 99 100 100 95 100 97 96 99 94 97 100 97 100 99 100 86 100 97 100 100 92 100 98 100 81 89 100 97 100 97 100 100
AJ 875393 KM 456196 KR 812232 KJ 735003 KJ710465 KM822751 JX380645 JX380645 KC965743 JF340251 HE814074 EU030400 AJ317958 JQ991868 KM232504 KT268950 HG328017 KP404098 KF493967 HQ832983 DQ865095 LN882435 DQ093725 EU520640 KP889853 FN689696 EU516991 KF385273 KF039897 EU479770 LN808976 KC551997 HM635337 GU054164 GQ179993 KF514671 KC222720 JF817335 GQ254683 FJ552729 KP235661 LN896693 JX421713 FN394724 KT878352 AJ269851 KP336756 GU055674 KT692593 FJ827743
17.6% 7.0% 3.7% 2.9% 2.1% 1.8% 1.6% 1.6% 1.6% 1.5% 1.5% 1.5% 1.2% 1.1% 1.1% 1.1% 1.0% 1.0% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6%
a b
Percent of sequences in Illumina sequencing library, genus which was rarely reported as deep-sea fungi, cmore than two OTUs belonged to the taxon, only one OTU number is shown in Table 2
Illumina sequencing library (Fig. 5). The result showed that Exophiala equina, Aspergillus pseudodeflectus, Penicillium sp. and Pyronemataceae sp. were the dominant phylotypes in sample B, while Leptosphaeria sp., Sporomiaceae sp., Coniella sp., Plectoshaerella citrullae, Faurelina indica dominated in sample D. Lecythophora fasciculate and Myriogenospora atramentosa were mainly found in samples A and C, respectively.
4. Discussion Although high-throughpu sequencing approach, the most successful and most widely adopted sequencing platform, had been widely applied in investigating fungal diversity of many environmental samples, such as soil (Kawahara et al., 2012), plants (Arfi et al., 2012) and marine corals (Amend et al., 2012), it was rarely applied in exploring fungal diversity of deep-sea sediment
samples. To obtain the maximum amount of fungal diversity, Illumina sequencing combined with clone library analysis and culture-dependent approach was applied in investigating the fungal community of deep-sea sediments from Okinawa Trough. Only 38 fungal OTUs (data no shown) and 21 phylotypes (data no shown) were recovered by clone library analysis and culture-dependent approach, respectively. While a surprisingly diverse assemblage of fungi (420 OTUs) were detected by Illumina sequencing in this study. Compared with previous studies on fungal diversity of deep-sea sediments by clone library analysis and culture-dependent approach (Nagano et al., 2010; Zhang et al., 2013 and 2014; Jebaraj et al., 2010; Zhang et al., 2015b), the present results suggested that high-throughpu Illumina sequencing could dramatically accelerate the discovery of fungal community of deep-sea sediments. The main reason may be that a large number of fungal sequences can be obtained easily by high-throughpu Illumina sequencing. To the best of our knowledge, this is the first exploration
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Fig. 3. Bray-Curtis analysis of dissimilarity of fungal communities between each two out of four deep-sea sediment samples (A–D) from Okinawa Trough.
Fig. 4. Principal component analysis (PCA) on the abundance of fungal phyla recovered from four deep-sea sediment samples (A–D) from Okinawa Trough by high–throughput Illumina sequencing. The scatter plot is of principal coordinate 1 (PC1) vs principal coordinate 2 (PC2). The percentages are the percentage of variation explained by the components.
of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughpu Illumina sequencing. Besides OTU-rich, the fungal community in deep-sea sediment from Okinawa Trough was taxonomically diverse. Our results showed that 170 fungal taxa belonged to four formally described fungal phyla and several unassigned phyla were recovered in this study. More than 12 taxa accounted for 21.5% sequences were found to be rarely recovered in deep-sea environments (Table 2), suggesting that the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments (Nagano et al., 2010; Damare et al., 2006; Zhang et al., 2014; Jebaraj et al., 2010; Nagahama and Nagano, 2012; Burgaud et al., 2009). Among the five Ascomycetous classes detected in this study, Sordariomycetes was the most diverse and abundant fungal class (Fig. 5 and Table 2), which challenged the traditional view that there were low diverse Sordariomycetes phylotypes in deep-sea environments (Nagahama et al., 2011). The most prevalent Sordariomycetous genera detected in this study were Myriogenospora, Faurelina, Lecythophora, Fusarium and Plectosphaerlla. Fusarium and Lecythophora could be frequently found in deep-sea hydrothermal vents and sediments (Zhang et al., 2013; Jebaraj et al., 2010; McGuire et al., 2013), while Faurelina, Myriogenospora and Plectosphaerella were rarely recovered in deep-sea environments
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in previous studies. In addition, despite accounted for a small percentage of the total sequences, three Ascomycetous OTUs affiliated with fungal genera Chaetomium, Pseudogymnoascus and Stemphylium could be found in all the four sediment samples in this study. In previous studies, Pseudogymnoascus and Stemphylium were rarely recovered from deep-sea environments. However, our results suggested that Pseudogymnoascus and Stemphylium could be distributed widely in deep-sea sediments from Okinawa Trough. The phylum Basidiomycota has been reported in high abundance in many deep-sea sediments (Xu et al., 2014 and 2016; Zhang et al., 2014 and 2016), but was relatively scarce in the deepsea sediments from Okinawa Trough. Zygomycota and Chytridiomycota were mainly found in two samples in this study (Fig. 2). Most sequences in Zygomycota and Chytridiomycota matched to genera Mortierella and Phlyctochytrium, respectively (Table 2), which had been reported as common fungal genera in soil and marine sediments (Zhang et al., 2013; Schmidt et al., 2008; Gryndler, 2000). Despite the important roles of many members of Zygomycota and Chytridiomycota in the soil or marine sediments (Jebaraj et al., 2010; Lozupone and Klein, 2002; Lim et al., 2010), little is known about their ecology or abundance in deep-sea environments. Compared analysis showed that 83–94% dissimilarity of fungal communities existed between each two samples (Fig. 3). This phenomenon is probably due to the difference in the locations and geochemical properties of different sediment samples. Eurotiomycetes and Pezizomycetes were mainly distributed in Iheya Ridge region (samples A and B), while Dothideomycetes, Leotiomycetes and Tremellomycetes were mainly detected in Iheya North region (samples C and D) (Fig. 5), likely as a result of environment difference between these two regions. Moreover, the richness of fungal communities in samples B and D were higher than that in samples A and C, respectively (Fig. 5 and Table 1), suggesting that the distance from active hydrothermal vent sites might be a significant impact on fungal communities (samples A and C were close from the active hydrothermal vents in Iheya North and Iheya Ridge respectively, while samples B and D were relatively distant from the vent sites). In addition, Fusarium, known as a C-fixing fungal genus in previous studies (Larmour and Marchant, 1977; Parkinson et al., 1991), was widely distributed in samples B, C and D in this study, suggesting Fusarium spp. might contribute to a relatively higher concentration of TOC in samples B, C and D than that in sample A. The Mn-oxidizing fungal communities (including Pleosporales and Pencillium) recognized as scavengers of Mn (Miyata et al., 2007), were more abundant in samples B and D than that in samples A and C, suggesting they could accumulate a higher concentration of Mn in samples B and D than that in samples A and C. These results suggested that various amounts of metals could be related to the differing fungal communities in the sediment samples. To conclude, this study reported a new insight into the fungal communities in deep-sea sediments from Okinawa Trough using high-throughpu Illumina sequencing. A total of 420 fungal OTUs and 170 taxa recovered from these sediment samples indicated that Illumina sequencing could dramatically accelerate the discovery of fungal community of deep-sea sediments. However, only one pair of primers was chosen to amplify the ITS1 sequences, suggesting a bias would be caused by specificity in this study. In future studies of the fungal diversity in deep-sea environments by Illumina sequencing, more primer pairs should be chosen to circumvent the biases caused by specificity. In addition, in order to obtain an even greater abundance of fungal communities in deepsea environments, it is necessary to combine Illumina sequencing with other methods, such as the microscopic observation of samples appropriately staining, measuring ergosterol,
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Fig. 5. Taxonomic composition profile of four deep-sea sediment samples (A–D) from Okinawa Trough analyzed based upon the 20 most abundant ITS1 (internal transcribed spacer 1) sequences in the Illumina sequencing library. The numbers after the taxonomic rank is the relative abundance of the corresponding taxon in the total ITS1 sequences in the Illumina sequencing library.
metagenomic methods, and other new powerful tools expected to be developed in the future (Zhang et al., 2014).
Acknowledgments We thank the research vessel KEXUE of the Chinese Academy of Sciences for collecting samples and WPOS sample center for providing samples. The authors are grateful for the financial support
provided by Strategic Leading Special Science and Technology Program of Chinese Academy of Sciences (XDA100304002), Natural Science Foundation of China (41206139, 41376160), Regional Innovation Demonstration Project of Guangdong Province Marine Economic Development (GD2012-D01-002), Science and Technology Project of Guangdong Province (2013B031100001), and Guangzhou Science and Technology Research Projects (201504291500134). The authors thank Dr. Zhao Jing (Novogene Bioinformatic Technology Co., Ltd, Beijing, China) for data analysis.
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Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing Xiao-Yong Zhang a,b,c, Guang-Hua Wang a,b,c, Xin-Ya Xu a,b,c, Xu-Hua Nong a,b,c, Jie Wang a,b,c, Muhammad Amin a,b,c, Shu-Hua Qi a,b,c,n a Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China b Guangdong Key Laboratory of Marine Material Medical, South China Sea Institute of Oceanology, Chinese Aacademy of Sciences, 164 West Xingang Road, Guangzhou 510301, China c RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China
art ic l e i nf o
a b s t r a c t
Article history: Received 18 May 2016 Received in revised form 6 August 2016 Accepted 16 August 2016 Available online 17 August 2016
The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deepsea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Fungal diversity Deep-sea sediments Okinawa Trough Illumina sequencing ITS1
1. Introduction Although there is only about 1% of the photosynthetically produced organic carbon that reaches to the deep-sea floor, these deep-sea sediments are now recognized as a home to rich and diverse microbial communities which provide a fundamental contribution to global carbon recycling through the sequestration and remineralization of organic matter (Jamieson et al., 2013). The presence and ecological importance of bacteria in deep-sea sediments have been well recognized ever since Zobell and Morita (1957) isolated bacteria specifically adapted to grow under high pressures and termed them “piezophile” (Whitman et al., 1998; Li et al., 1999; Sogin et al., 2006). However, the fungi in deep-sea n Corresponding author at: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301, China. E-mail address: [email protected] (S.-H. Qi).
http://dx.doi.org/10.1016/j.dsr.2016.08.004 0967-0637/& 2016 Elsevier Ltd. All rights reserved.
sediments have been neglected previously. One of the first reports of fungi in deep-sea sediments was provided by Kohlmeyer and Kohlmeyer (1979), who reported obligate marine fungi from wooden panels immersed at depths of 500–3000 m. Since then, fungi have been found in different deep-sea sediments from the Mariana Trench (Takami et al., 1997; Nagano et al., 2010), Chagos Trench (Raghukumar et al., 2004), Indian Ocean (Damare et al., 2006; Singh et al., 2011 and 2012; Zhang et al., 2014), South China Sea (Lai et al., 2007; Zhang et al., 2013), Peru Trench (Edgcomb et al., 2011), Gulf of Mexico (Thaler et al., 2012) and Pacific Ocean (Xu et al., 2014 and 2016). Despite of the recent advances, the knowledge and understanding pertaining to the diversity and distribution of fungal communities in deep-sea sediments is still limited. The early studies on fungal diversity in deep-sea sediments were mainly based on cultivation techniques (Damare et al., 2006; Le Calvez et al., 2009; Jebaraj et al., 2010). Fungal species detected in deep-sea sediments by culture-dependent methods mostly
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belonged to the phylum Ascomycota and limited yeast species of Basidiomycota (Nagano and Nagahama, 2012). However, the traditional molecular approach (clone library analysis) indicated that cultivable fungi were only a small fraction of the total fungi in deep-sea sediments (Nagano et al., 2010). Recently, an increasing number of novel fungal phylotypes including DSF-group and BCGI clade (Damare et al., 2006), KML11 clade and Rozella (Burgaud et al., 2009) have been detected by clone library analysis. However, this technique of clone library analysis represents a low throughput, costly and tedious method, providing only limited information of the diversity associated with the number of clones analyzed (Monchy et al., 2011). With the recent development of DNA sequencing technologies, high-throughpu sequencing of rRNA gene fragments has been introduced to the field of fungal ecology, which can detect much deeper fungal community in diverse environments, such as soil (Buee et al., 2009) and phyllosphere (Jumpponen and Jones, 2009; Jumpponen et al., 2010). In this study, we presented the use of high-throughpu Illumina sequencing to assess the fungal diversity in deep-sea sediments from Okinawa Trough.
2. Materials and methods 2.1. Study site and sampling Four deep-sea sediment samples (A–D) were collected using electro hydraulic grab with underwater television camera (for samples A and C) and box sampler (for samples B and D) during the Okinawa Trough Cruise. Latitudes and longitudes, and depths of the collected deep-sea sediment samples A–D were 27°48.47'N and 126°54.32'E, 1190 m; 27°48.12'N and 126°58.89'E, 1330 m; 27°33.07'N and 126°58.36'E, 1387 m; 27°34.01'N and 126°55.59'E, 1589 m; respectively (Zhang et al., 2015a). The collected sediment samples were mostly undisturbed and compact. The average length of the sediment cores collected from these locations was approximately 30 cm. Subsections of 3 cm down to 8 cm were cut from the sediment sub-cores and immediately stored in sterile plastic bags to avoid any aerial contamination. The bags were sealed and stored at –20 °C until processing of direct DNA extraction (Zhang et al., 2014). The distances of samples A and B from the active hydrothermal vents in Iheya North were about 1.1 km and 6.3 km, respectively. While the distances of samples C and D from the active hydrothermal vents in Iheya Ridge were about 0.4 km and 4.7 km, respectively. The geochemical properties (including total organic carbon (TOC), total nitrogen (TN), total sulfur (TS), and several metals) of the four sediment samples were described by Zhang et al. (2015a). 2.2. DNA extraction and Illumina sequencing Total genomic DNA from two grams of sediment samples was extracted directly using the FastDNAs spin kit (MP bio, Santa Ana, USA) according to the manufacturer's instruction. DNA concentration was then determined using a NanoDrop ND-2000 UV– vis spectrophotometer (Thermo Scientific, Wilmington, USA). According to the concentration, DNA was diluted to 1 ng/μL using sterile water. Total genomic DNA was amplified using the primers ITS1F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2 (5′GCTGCGTTCTTCATCGATGC-3′) that amplified the internal transcribed spacer-1 (ITS1) region of the fungal rRNA gene (White et al., 1990). PCR reactions were carried out in 30 μL reactions with 15 μL of Phusions High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of forward and reverse primers, and about 10 ng templates DNA. Thermal cycling consisted of initial denaturation
at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and elongation at 72 °C for 2 min, and a final extension at 72 °C for 10 min. The presence of PCR products was determined by analyzing 3 μL of product on 2% agarose gel. Samples with bright main strip between 100 and 500 bp were chosen for further experiments. PCR products were mixed in equidensity ratios. Then, mixture of PCR products were purified with GeneJET Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using NEB Nexts Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the [email protected] Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. At last, the library was sequenced on an Illumina MiSeq platform (Version 3) at Novogene Bioinformatic Technology Co., Ltd, Beijing, China. 2.3. OTU cluster and species annotation The raw Illumina sequences were assigned to individual samples based on their unique barcodes. The barcodes and ITS1 primer sequence were then removed to generate Paired-end reads (about 250 bp). Raw tags were then generated by merging Paired-end reads with FLASH, a very fast and accurate analysis tool (Magoc and Salzberg, 2011). The raw tags were quality filtered and analyzed with the Quantitative Insight Into Microbial Ecology (QIIME) software package (Caporaso et al., 2010). Operational taxonomic units (OTUs) were assigned using QIIME's Uparse v7.0.1001 with a threshold of 97% pairwise identity (Edgar, 2013). OTUs were classified taxonomically using QIIME-based wrapper of the Ribosomal Database Project (RDP) classifier (Wang et al., 2007) against the UNITE database (http://unite.ut.ee/, Version 2014.05.13). Any OTUs representing non-fungal sequences were removed before downstream analysis (Bokulich et al., 2013). 2.4. Diversity analysis OTUs abundance information was normalized using a standard of sequence number corresponding to the sample with the least sequences (Gihring et al., 2011). Subsequent analysis of alpha diversity (with-in sample species richness) and beta diversity (between-sample community dissimilarity) was performed basing on this output normalized data. In order to reveal alpha diversity, we rarified the OTU table and calculate three metrics: Chao1 estimates the species abundance; Observed Species estimates the amount of unique OTUs found in each sample, and Shannon's index provides a means of comparing the diversity between two or more samples (Schloss et al., 2009). Rarefaction curves were generated based on these three metrics. In beta diversity analysis, cluster analysis was preceded by principal component analysis (PCA) (Jolliffe, 1986) and Bray-Curtis dissimilarity (Bray and Curtis, 1957) was calculated using the QIIME software package. 2.5. Nucleotide sequence accession numbers Raw sequences of samples A–D were deposited in the Sequence Read Archives of the NCBI under accession number SRR 3234033– 3234036.
3. Results 3.1. Illumina sequencing and sequence analysis After denoising and chimera detection, a total of 41,801 sequences were obtained. After removed 1504 non-fungal sequences
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diversity indices (H′) were also calculated, ranging from 1.2 to 6.1. 3.2. Fungal community composition
Fig. 1. Rarefaction curves constructed for fungal ITS1 (internal transcribed spacer 1) sequences recovered from four deep-sea sediment samples (A–D) from Okinawa Trough by high-throughpu Illumina sequencing. Table 1 Comparison of the estimated operational taxonomic unit (OTU) richness, diversity indexes of the fungal ITS1 sequences for clustering at 97% sequences similarity from the sequencing analysis. Sample Name
Observed OTU
Chao1
Shannon
A B C D
37 112 139 241
44.273 184.565 158.029 310.400
1.613 2.819 4.050 6.122
(consisting of plant-associated sequences), a total of 40,297 fungal ITS1 sequences were retained and used for analysis, which were clustered into 420 OTUs with 97% sequence similarity. The numbers of fungal ITS1 sequences and OTUs detected in samples A–D were 3324 and 37, 17872 and 112, 3352 and 139, and 15749 and 241, respectively. Rarefaction curves were constructed for the number of ITS1 sequences vs OTUs (Fig. 1). Only sample A demonstrated a plateau, indicating that the number of sequences analyzed might sufficiently represent the fungal diversity in the sample. This result was confirmed by a comparison of the observed and estimated OTUs (Chao 1 indices) at the 97% sequence similarity level. With the Chao 1 indices, the numbers of observed OTUs were close to those estimated for samples A and C. While for the other two samples (B and D), the numbers of observed OTUs were lower than the estimated OTUs (Table 1). The Shanon's
A total of 170 fungal taxa detected in four deep-sea sediment samples covered a wide variety of organisms from four formally described fungal phyla and several unassigned fungal phyla. About 78% of the total fungal sequences belonged to Ascomycota, followed by Basidiomycota (17.3%), and a small proportion belonged to Zygomycota (1.5%), Chytridiomycota (0.8%) and several unassigned fungal phyla (2.4%) (Fig. 2). These taxa were mainly distributed in eight fungal classes: Sordariomycetes (46%), Dothideomycetes (8%), Eurotiomycetes (6%), Leotiomycetes (3%), Pezizomycetes (2%), Agaricomycetes (7%), Tremellomycetes (2%) and Chytridiomycetes (0.7%) (Table 2). Among the 50 most abundant fungal taxa (Table 2), 17 taxa (31.7%) could be identified as unassigned fungal genera in the UNITE database (http://unite.ut.ee/, Version 2014.05.13); and 21 taxa (20.2%) were common fungal genera frequently found in different deep-sea environments (Nagano et al., 2010; Damare et al., 2006; Zhang et al., 2014; Jebaraj et al., 2010; Nagahama and Nagano, 2012; Burgaud et al., 2009), while the remaining 12 taxa (21.5%) were rarely recovered in deep-sea environments (Table 2). 3.3. Comparison of the fungal community structures in different samples The Bray-Curtis analysis showed 83–94% dissimilarity of fungal communities between each two sediment samples (Fig. 3). There were 14, 66, 32 and 132 unique OTUs in samples A, B, C and D, respectively. Only 3 OTUs affiliated with fungal genera Pseudogymnoascus, Stemphylium and Chaetomium, were shared among the four samples. The principal component analysis clearly showed variations in fungal community among different sediment samples (Fig. 4). Comparative analysis of the fungal community structures in the four sediment samples revealed a distinct distribution of fungal phyla (Fig. 2). For instance, Ascomycota accounted for 75–95% of the total sequences in samples A, B and D, whereas this phylum accounted for only 49% of the total sequences in sample C (Fig. 2). Alternatively, sample C was characterized by a relatively higher percentage of Basidiomycetous fungi compared with the other samples. In addition, Zygomycota was mainly found in samples C and D, while Chytridiomycota was recovered in samples B and D. To further compare the distinct communities of the four samples, taxonomic composition profile of different four samples were analyzed based upon the 20 most abundant ITS1 sequences in the
Fig. 2. Relative abundance of the fungal phyla recovered from four deep-sea sediment samples (A–D) from Okinawa Trough by high-throughpu Illumina sequencing.
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Table 2 The 50 most abundant fungal taxa (accounted for 73.4%) in the Illumina sequencing library. No. taxon
Phylum
Class
Genus
No. OTU
Similarity %
GenBank accession
Totala
1c 2c 3 4 5 6 7 8 9 10 11c 12 13c 14 15c 16 17c 18c 19 20 21c 22 23 24 25 26 27 28 29 30c 31 32 33 34 35 36 37 38c 39 40 41 42 43 44c 45c 46c 47c 48c 49 50
Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Basidiomycota Basidiomycota Ascomycota Basidiomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota unassigned Zygomycota Ascomycota unassigned Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Basidiomycota Basidiomycota Ascomycota Ascomycota Ascomycota Basidiomycota Zygomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Ascomycota Chytridiomycota
Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Leotiomycetes Sordariomycetes Pezizomycetes unassigned Sordariomycetes Agaricomycetes Tremellomycetes Dothideomycetes Agaricomycetes Eurotiomycetes Sordariomycetes Eurotiomycetes Eurotiomycetes Dothideomycetes Eurotiomycetes Dothideomycetes unassigned incertaesedis Dothideomycetes unassigned Sordariomycetes Dothideomycetes Sordariomycetes Dothideomycetes Sordariomycetes Agaricomycetes Agaricomycetes Dothideomycetes Sordariomycetes Sordariomycetes Agaricomycetes incertaesedis Dothideomycetes Dothideomycetes Eurotiomycetes Eurotiomycetes Dothideomycetes Leotiomycetes incertaesedis Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Chytridiomycetes
unassigned Myriogenosporab Faurelinab Lecythophora Coniellab Thelebolusb Fusarium unassigned unassigned Plectosphaerellab Russulab unassigned Leptosphaeria unassigned Aspergillus Fusarium Exophiala Penicillium unassigned Paecilomyces Preussiab unassigned Mortierella unassigned unassigned unassigned unassigned unassigned Pseudogymnoascusb Myrotheciumb Schizophyllum unassigned Corynespora unassigned Trichocladium unassigned Mortierella Phoma Lophiostoma Emericella unassigned Stemphyliumb unassigned Minimidochiumb Acremonium Nectria Chaetomium Podospora Microdochium Phlyctochytriumb
300 5 33 286 3 10 55 9 11 7 275 106 180 186 170 101 234 81 112 43 23 111 154 31 45 83 37 155 51 314 87 42 66 20 12 378 60 53 82 363 450 80 48 160 70 49 223 100 92 41
97 98 100 100 100 100 100 98 89 100 100 100 100 96 100 100 100 100 99 100 100 95 100 97 96 99 94 97 100 97 100 99 100 86 100 97 100 100 92 100 98 100 81 89 100 97 100 97 100 100
AJ 875393 KM 456196 KR 812232 KJ 735003 KJ710465 KM822751 JX380645 JX380645 KC965743 JF340251 HE814074 EU030400 AJ317958 JQ991868 KM232504 KT268950 HG328017 KP404098 KF493967 HQ832983 DQ865095 LN882435 DQ093725 EU520640 KP889853 FN689696 EU516991 KF385273 KF039897 EU479770 LN808976 KC551997 HM635337 GU054164 GQ179993 KF514671 KC222720 JF817335 GQ254683 FJ552729 KP235661 LN896693 JX421713 FN394724 KT878352 AJ269851 KP336756 GU055674 KT692593 FJ827743
17.6% 7.0% 3.7% 2.9% 2.1% 1.8% 1.6% 1.6% 1.6% 1.5% 1.5% 1.5% 1.2% 1.1% 1.1% 1.1% 1.0% 1.0% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.7% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6%
a b
Percent of sequences in Illumina sequencing library, genus which was rarely reported as deep-sea fungi, cmore than two OTUs belonged to the taxon, only one OTU number is shown in Table 2
Illumina sequencing library (Fig. 5). The result showed that Exophiala equina, Aspergillus pseudodeflectus, Penicillium sp. and Pyronemataceae sp. were the dominant phylotypes in sample B, while Leptosphaeria sp., Sporomiaceae sp., Coniella sp., Plectoshaerella citrullae, Faurelina indica dominated in sample D. Lecythophora fasciculate and Myriogenospora atramentosa were mainly found in samples A and C, respectively.
4. Discussion Although high-throughpu sequencing approach, the most successful and most widely adopted sequencing platform, had been widely applied in investigating fungal diversity of many environmental samples, such as soil (Kawahara et al., 2012), plants (Arfi et al., 2012) and marine corals (Amend et al., 2012), it was rarely applied in exploring fungal diversity of deep-sea sediment
samples. To obtain the maximum amount of fungal diversity, Illumina sequencing combined with clone library analysis and culture-dependent approach was applied in investigating the fungal community of deep-sea sediments from Okinawa Trough. Only 38 fungal OTUs (data no shown) and 21 phylotypes (data no shown) were recovered by clone library analysis and culture-dependent approach, respectively. While a surprisingly diverse assemblage of fungi (420 OTUs) were detected by Illumina sequencing in this study. Compared with previous studies on fungal diversity of deep-sea sediments by clone library analysis and culture-dependent approach (Nagano et al., 2010; Zhang et al., 2013 and 2014; Jebaraj et al., 2010; Zhang et al., 2015b), the present results suggested that high-throughpu Illumina sequencing could dramatically accelerate the discovery of fungal community of deep-sea sediments. The main reason may be that a large number of fungal sequences can be obtained easily by high-throughpu Illumina sequencing. To the best of our knowledge, this is the first exploration
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Fig. 3. Bray-Curtis analysis of dissimilarity of fungal communities between each two out of four deep-sea sediment samples (A–D) from Okinawa Trough.
Fig. 4. Principal component analysis (PCA) on the abundance of fungal phyla recovered from four deep-sea sediment samples (A–D) from Okinawa Trough by high–throughput Illumina sequencing. The scatter plot is of principal coordinate 1 (PC1) vs principal coordinate 2 (PC2). The percentages are the percentage of variation explained by the components.
of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughpu Illumina sequencing. Besides OTU-rich, the fungal community in deep-sea sediment from Okinawa Trough was taxonomically diverse. Our results showed that 170 fungal taxa belonged to four formally described fungal phyla and several unassigned phyla were recovered in this study. More than 12 taxa accounted for 21.5% sequences were found to be rarely recovered in deep-sea environments (Table 2), suggesting that the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments (Nagano et al., 2010; Damare et al., 2006; Zhang et al., 2014; Jebaraj et al., 2010; Nagahama and Nagano, 2012; Burgaud et al., 2009). Among the five Ascomycetous classes detected in this study, Sordariomycetes was the most diverse and abundant fungal class (Fig. 5 and Table 2), which challenged the traditional view that there were low diverse Sordariomycetes phylotypes in deep-sea environments (Nagahama et al., 2011). The most prevalent Sordariomycetous genera detected in this study were Myriogenospora, Faurelina, Lecythophora, Fusarium and Plectosphaerlla. Fusarium and Lecythophora could be frequently found in deep-sea hydrothermal vents and sediments (Zhang et al., 2013; Jebaraj et al., 2010; McGuire et al., 2013), while Faurelina, Myriogenospora and Plectosphaerella were rarely recovered in deep-sea environments
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in previous studies. In addition, despite accounted for a small percentage of the total sequences, three Ascomycetous OTUs affiliated with fungal genera Chaetomium, Pseudogymnoascus and Stemphylium could be found in all the four sediment samples in this study. In previous studies, Pseudogymnoascus and Stemphylium were rarely recovered from deep-sea environments. However, our results suggested that Pseudogymnoascus and Stemphylium could be distributed widely in deep-sea sediments from Okinawa Trough. The phylum Basidiomycota has been reported in high abundance in many deep-sea sediments (Xu et al., 2014 and 2016; Zhang et al., 2014 and 2016), but was relatively scarce in the deepsea sediments from Okinawa Trough. Zygomycota and Chytridiomycota were mainly found in two samples in this study (Fig. 2). Most sequences in Zygomycota and Chytridiomycota matched to genera Mortierella and Phlyctochytrium, respectively (Table 2), which had been reported as common fungal genera in soil and marine sediments (Zhang et al., 2013; Schmidt et al., 2008; Gryndler, 2000). Despite the important roles of many members of Zygomycota and Chytridiomycota in the soil or marine sediments (Jebaraj et al., 2010; Lozupone and Klein, 2002; Lim et al., 2010), little is known about their ecology or abundance in deep-sea environments. Compared analysis showed that 83–94% dissimilarity of fungal communities existed between each two samples (Fig. 3). This phenomenon is probably due to the difference in the locations and geochemical properties of different sediment samples. Eurotiomycetes and Pezizomycetes were mainly distributed in Iheya Ridge region (samples A and B), while Dothideomycetes, Leotiomycetes and Tremellomycetes were mainly detected in Iheya North region (samples C and D) (Fig. 5), likely as a result of environment difference between these two regions. Moreover, the richness of fungal communities in samples B and D were higher than that in samples A and C, respectively (Fig. 5 and Table 1), suggesting that the distance from active hydrothermal vent sites might be a significant impact on fungal communities (samples A and C were close from the active hydrothermal vents in Iheya North and Iheya Ridge respectively, while samples B and D were relatively distant from the vent sites). In addition, Fusarium, known as a C-fixing fungal genus in previous studies (Larmour and Marchant, 1977; Parkinson et al., 1991), was widely distributed in samples B, C and D in this study, suggesting Fusarium spp. might contribute to a relatively higher concentration of TOC in samples B, C and D than that in sample A. The Mn-oxidizing fungal communities (including Pleosporales and Pencillium) recognized as scavengers of Mn (Miyata et al., 2007), were more abundant in samples B and D than that in samples A and C, suggesting they could accumulate a higher concentration of Mn in samples B and D than that in samples A and C. These results suggested that various amounts of metals could be related to the differing fungal communities in the sediment samples. To conclude, this study reported a new insight into the fungal communities in deep-sea sediments from Okinawa Trough using high-throughpu Illumina sequencing. A total of 420 fungal OTUs and 170 taxa recovered from these sediment samples indicated that Illumina sequencing could dramatically accelerate the discovery of fungal community of deep-sea sediments. However, only one pair of primers was chosen to amplify the ITS1 sequences, suggesting a bias would be caused by specificity in this study. In future studies of the fungal diversity in deep-sea environments by Illumina sequencing, more primer pairs should be chosen to circumvent the biases caused by specificity. In addition, in order to obtain an even greater abundance of fungal communities in deepsea environments, it is necessary to combine Illumina sequencing with other methods, such as the microscopic observation of samples appropriately staining, measuring ergosterol,
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Fig. 5. Taxonomic composition profile of four deep-sea sediment samples (A–D) from Okinawa Trough analyzed based upon the 20 most abundant ITS1 (internal transcribed spacer 1) sequences in the Illumina sequencing library. The numbers after the taxonomic rank is the relative abundance of the corresponding taxon in the total ITS1 sequences in the Illumina sequencing library.
metagenomic methods, and other new powerful tools expected to be developed in the future (Zhang et al., 2014).
Acknowledgments We thank the research vessel KEXUE of the Chinese Academy of Sciences for collecting samples and WPOS sample center for providing samples. The authors are grateful for the financial support
provided by Strategic Leading Special Science and Technology Program of Chinese Academy of Sciences (XDA100304002), Natural Science Foundation of China (41206139, 41376160), Regional Innovation Demonstration Project of Guangdong Province Marine Economic Development (GD2012-D01-002), Science and Technology Project of Guangdong Province (2013B031100001), and Guangzhou Science and Technology Research Projects (201504291500134). The authors thank Dr. Zhao Jing (Novogene Bioinformatic Technology Co., Ltd, Beijing, China) for data analysis.
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