Metagenomics Study of Fungi and Fungi-Like Organisms Associated With the Seagrass Halophila Stipulacea (Forssk.) Asch. From Al-Leith Mangroves, Saudi Arabia

This is the rst metagenomics study of the fungal community associated with leaves of the seagrass Halophila stipulacea. Five leave samples were collected from the Al-Leith mangroves along the Red Sea coast of Saudi Arabia. Total DNA was extracted from 250 mg of each sample and the rst 300 bp (contains D1-D2 variable regions) of the LSU rDNA amplicon was sequenced with the Illumina MiSeq (bTEFAP). A total of 928,626 reads were obtained from the ve samples. The sequence reads belonged to the kingdoms: Metazoa (48.1% of the total reads), Viridiplantae (41.1%), Eukaryota (8.8%), Fungi (1.96%), Bacteria (0.09%), and Archaea (0.0001%). Fungi represented between 1.1% and 5.8% of the total reads in the ve samples. A total of 18,279 reads (representing 1.96% of the total reads) were recorded from the 5 samples representing 296 molecular species (OTUs) that belong to 13 fungal phyla. At the phylum level, Basidiomycota dominated the community (37.2–51.6%) in three samples, while Neocallimastigomycota (37.5%) and Mucoromycota (42.1%) dominated the community in the fourth and the fth sample respectively. High diversity of OTUs (28 molecular species) were recorded from the monokaryotic subkingdom with ve unknown basal lineages that are not aligning with any known taxa. Total number of sequence reads of fungi-like organisms (Stramenopiles) from the ve samples ranged between 0.16% of total reads in the fth sample (AL-Hs05) to 2.9% in the rst one (AL-Hs01). Majority of the fungi-like organisms reads (93.6%) belong to the phylum Oomycota, followed by Opisthokonts (Fungi/Metazoa group) representing 6.4% of fungi-like reads. Neocallimastigomycota) and fungi-like organisms occupied a major portion of the sequences reads followed by Basidiomycota and Ascomycota. Our results support ndings that the majority of fungi and fungi-like organisms’ communities are so far unknown with seven deep branching lineages remain to be cultivated.


Introduction
Seagrasses are owering plants inhabiting marine environments and have a worldwide distribution, with 50 species reported that cover about 60,000 km 2 (Logan 1992;Phang 2000;Raghukumar 2017). Seagrasses provide food, habitats, and breeding grounds to a variety of marine species. In addition, meadows of seagrass are important carbon sinks and sequester between 10 to 18% of the ocean's carbon reservoir for long-term storage (Pernice et al. 2016). Seagrasses prevent erosion by trapping and binding sediments and by their death, provide substantial amounts of nutrition to organisms living within the seagrass ecosystem, as well as to those in mangroves and corals and other ecosystems (Wilson 1998; Phang 2000; Raghukumar 2017). Primary productivity of seagrass meadows is among the highest of aquatic ecosystems (Duarte and Chiscano 1999). More than 50% of seagrass production enters the detrital food web (Duarte and Cebrian 1996).
Seagrasses represent one of the most valuable ecosystems on Earth, with an estimated value of $ 2.8 10 6 /yr/km 2 (Costanza et al. 2014).

Endophytic fungi of seagrasses
This study aims to document the fungal community from decaying leaves of the seagrass Halophila stipulacea using metagenomics study based on LSU rDNA.

Phylogenetic trees
We placed core OTUs into phylogenetic trees to investigate their relationships and re ne taxonomic assignments. OTUs were blasted in the GenBank and sequences with less than 95% similarity and unknowns were excluded from the analyses..
The most closely related sequences and the sequences of representative fungal species were aligned with our sequences by using CLUSTALX (Thompson et al. 1997

Phylogenetic analyses of OTUs referred to Ascomycota
Ten OTUs grouped with genera in the order Saccharomycetales with moderate statistical support (58 ML/ 78 MP/ 84 BYPP), of which six OTUs have phylogenetic a nity with the genus Pichia E.C. Hansen, two OTUs with Hanseniaspora Zikes, one with Yamadazyma Billon-Grand, and one with Cyberlindnera Minter. Yeasts were frequently recorded from mangroves, with more than 138 species referred to Ascomycota and 75 taxa to Basidiomycota that were recorded from OTUs nested within the order Hypocreales that include one OTU related to Fusarium Link with high statistical support (100 ML/ 100 MP/ 100 BYPP) and one OUT grouped with Trichoderma Pers. in a highly supported clade (Fig. 3).

Phylogenetic analyses of OTUs referred to Basidiomycota
Six OTUs nested within the clade of the yeast genus Malassezia Baill. with high statistical support (92 ML/ 94 MP/ 100 BYPP). The genus Malassezia represented 11.11% of the total fungal reads in the current study. The sequences reads of Malassezia were frequently recorded in high percentages in previous metagenomics studies from marine habitats (e.g.   Cuomo et al. 1985). Fungi-like organisms, monokaryon phyla and Basidiomycota are well represented in the current metagenomics study with a wider range of taxa than those recorded by culture-based methods. However, we need to be careful when interpreting the metagenomics data as the primers and methodology might bias the obtained results.
In the current study, seven deep branching lineages that did not cluster with any known microbial taxa were recorded which clearly shows that a wide range of microbes are yet to be cultured from the sea grass Halophila stipulacea. In a previous study of the bacterial community of H. stipulacea from Gulf of Elat, Red Sea based on 16S rDNA clone library (Weidner et al. 2000), they reported three unknown deep branching lineages that are not aligned with any known taxa. In summary, this study adds new knowledge of fungal diversity from decaying fronds of H. stipulacea and highlights a high diversity and abundance of fungi in the ve samples investigated and previously not known. While the number of marine fungi documented (1,901: www.marinefungi.org), largely due to direct observation of substrates and culture-based studies is impressive, metagenomic studies and the development and analysis of sequence data will greatly add to our knowledge and lead to a better understanding of their physiological and biochemical role in the marine environment. Clearly the many new linages highlighted in this study require further investigation, especially their identity, host and function.

Figure 2
Percentages of the phyla recorded from the ve metagenomics samples of Halophila stipulacea.

Figure 3
Maximum likelihood phylogenetic tree based on LSU rDNA of OTUs belong to Ascomycota and related sequences retrieved from the GenBank. The tree is rooted with representatives of Basidiomycota and Entorrhizomycota. Bootstrap support on the nodes represents ML and MP ≥50%. Branches with a BYPP of ≥95% are in bold. The sequences generated in this study are in red.

Figure 5
Maximum likelihood phylogenetic tree based on LSU rDNA of OTUs belong to monokaryon phyla and related sequences retrieved from the GenBank. The tree is rooted with representatives of Entorrhizomycota. Bootstrap support on the nodes represents ML and MP ≥50%. Branches with a BYPP of ≥95% are in bold. The sequences generated in this study are in red.