Genome Sequencing and analyses of Two Marine Fungi from the North Sea Unraveled a Plethora of Novel Biosynthetic Gene Clusters

Marine Fungi are potent secondary metabolite producers. However, limited genetic information are available their biosynthetic gene clusters (BGCs) and their biotechnological applications. To overcome this lack of information, herein, we used next-generation sequencing methods for genome sequencing of two marine fungi, isolated from the German Wadden Sea, namely Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079. The assembled genome size of the marine isolate Calcarisporium sp. KF525 is about 36.8 Mb with 60 BGCs, while Pestalotiopsis sp. KF079 has a genome size of 47.5 Mb harboring 67 BGCs. Of all BGCs, 98% and 97% are novel clusters of Calcarisporium sp. and Pestalotiopsis sp., respectively. Only few of the BGCs were found to be expressed under laboratory conditions by RNA-seq analysis. The vast majority of all BGCs were found to be novel and unique for these two marine fungi. Along with a description of the identified gene clusters, we furthermore present important genomic features and life-style properties of these two fungi. The two novel fungal genomes provide a plethora of new BGCs, which may have biotechnological applications in the future, for example as novel drugs. The genomic characterizations will provide assistance in future genetics and genomic analyses of marine fungi.

water salinity, the water movement, the presence of suitable substrates for colonization, the presence of propagates in the water, interspecific competition, pollution and the oxygen content of the water 7 .
The biodiversity of marine fungal isolates is mirrored by the molecular diversity of their secondary metabolites [8][9][10][11][12] . Yet, these studies are mainly chemistry based, and marine fungi remain tremendously underexplored with regard to species, distribution and applications 12, 13 . As such marine-derived fungi contain a treasure chest of secondary metabolites, of which a considerable number have promising biological or pharmaceutical properties 14 . However, so far, most studies did not use whole genome sequencing to discover the enormous potential of fungi for secondary metabolite production. A study on a marine fungus isolated from a sponge revealed the entire secondary metabolite cluster for scopularide A and B, which has anti-cancer activities 15 . Secondary metabolites are low-molecular-mass organic compounds that, unlike primary metabolites, are not directly involved in growth, development or reproduction of the producing organism. Up until 2014 about 170,000 natural products have been characterized from both marine and terrestrial organisms 16 . Fungal secondary metabolites can be divided into four main chemical classes: polyketides, terpenoids, shikimic acid-derived compounds, and non-ribosomal peptides. The majority of fungal secondary metabolites derives from either non-ribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs), A few compounds represent mixed polyketide-non-ribosomal peptide compounds called NRPS-PKS hybrids 17 . Furthermore, secondary metabolites can be found in microbes of diverse environments and even chemical biogeographic distribution maps for biomedically valuable families of natural products in the environment have been created 18 . A number of these compounds have important pharmacological applications and are used as antibiotics/antibacterial drugs 19 . Genome-mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated, because many of these gene clusters are silent under standard cultivation conditions 19 . This indicated a plethora of natural products remains to be discovered.
Here we report on the genomic sequences of two North Sea-derived fungal isolates, Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079 by the use of two next-generation sequencing methods. The marine-derived Calcarisporium sp. KF525 has a 36.8 Mb genome with 60 biosynthetic gene clusters (BGCs) and Pestalotiopsis sp. KF079 has a genome size of ~47.5 Mb and harbors 67 BGCs. The majority of these clusters has not been previously identified and hence provide a great source of new potential natural products. Here we make a first attempt to characterize the genome content of these two marine fungi and their BGCs using high-throughput methods.

Results
Overview of genomic features and RNA-Seq statistics. We generated hybrid genome assemblies of two marine fungi using two different small read sequencing methods, Roche 454 and Illumina HiSeq. 2000. The estimated genome sizes for these marine strains of Calcarisporium KF525 and Pestalotiopsis KF079 are about 36.8 Mb and about 47.5 Mb, respectively (Table 1). Genomic coverages for Calcarisporium sp. KF0525 and Pestalotiopsis KF079 and are 11.5× and 9.3× using Roche 454 reads and 219× and 169× using Illumina HiSeq 2000 reads, respectively ( Table 1). The estimated numbers of genes are 15,459 and 22,626, respectively (Table 1 and Figure S1). These data are within the range of genome sizes and corresponding gene contents of ascomycetes, which are rather variable in both land and marine fungi ( Figure S1). The genome assembly is simplified by the fact that both Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079 possess a limited number of repeated DNA sequences, which account for 1.28% and 0.97% of their respective genome sizes only (Table S1). Using homology-based genome annotation analyses, we were able to annotate 72% (Table S2) and 68.9% (Table S3) of encoded proteins from assembled genomes of Calcarisporium KF525 and Pestalotiopsis KF079, respectively.
We performed RNA-Seq with wild-type strains of Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079 and we found a low number of only 4,401 and 6,658 genes, being expressed under laboratory conditions. This accounts for 28.5% and 29.43% of total genes in the corresponding genomes (Tables 2, S4-S5). Based on the reads per kilobase of transcript per million mapped reads (RPKM) values, we divided the expressed genes into nine tier (Tables 2, S4-S5).
Phylogenomic relationships of the two marine fungi. Genome-wide annotations of Calcarisporium sp. and Pestalotiopsis sp. depicted that both marine fungi are members of the Sordariomycetes, and they are most closely related to Nectria and Fusarium species ( Figure S2A). We also performed a phylogenomic analysis using the CVtree 22 , which revealed that Calcarisporium sp. is close to Metarhizium and Trichoderma. In contrast, Pestalotiopsis sp. has diverged early from other representative sordariomycetic fungi ( Figure S2B) and is close to another marine fungus, S. brevicaulis 15 . Overview of protein domains in the two marine fungi. Independently foldable structural protein units serve as protein domains, which share common functions that have been conserved during evolution. Generally, these protein domains are surveyed in newly sequenced genomes using two databases -Pfam (version 27 23 ) and Interpro (version 43 24 ). Calcarisporium sp. and Pestalotiopsis sp. genomes encode 2,599 and 3,613 different protein domains localized in 7,272 (Table S6) and 10,383 proteins (Table S7), respectively. The top 20 Pfam domains are summarized in Fig. 1. We identified 1,317 and 1,231 WD domain, G-beta repeat (WD40, Pfam ID -PF00400) in Calcarisporium sp. and Pestalotiopsis sp. genomes, respectively. WD40 domains are involved in the signal transduction and regulate fungal cell differentiation processes 25 . There were furthermore a total of 1,711 and 1,566 Ankyrin repeats (Ank, PF00023) in Calcarisporium sp. and Pestalotiopsis sp. genomes, respectively. Ankyrin repeats consists of 30-34 amino acid residues long protein motifs and assist protein-protein interactions 26 . Moreover, Calcarisporium sp. genome encodes two tetratricopeptide-repeat-carrying domains (TPR_1, PF00515 and TPR_2, PF07719), which are found in 418 TPR_1 and 569 TPR_2 proteins, while the Pestalotiopsis sp. genome contains 450 TPR_1 and 610 TPR_2 proteins. These TPR motifs function as protein interaction modules 27  seven terpene and two heterocyst-type glycolipid ketosynthase (Hglks) clusters. In addition, there are four hybrid clusters and seven putative clusters. Hybrid clusters include two NRPS-t1PKS and one each of Hglks-t1PKS and T1PKS-NRPS (Table S8). 59 BGCs are unique to Calcarisporium sp., as these BGCs have no homologs clusters in the MiBiG database ( 28 a database of known BGCs). Hence, more than 98% of detected BGCs of Calcarisporium sp. are by and large novel with some identities to uncharacterized BGCs in different species. Only one cluster, mCaBGC19, is known and has a homolog in the trichotecene gene cluster from Fusarium in the MiBiG database 28 , which is the database of known and characterized BGC.

PKS clusters in Calcarisporium sp. All the PKS genes and their expression based (on RNA-seq data) in
standard growth media are shown in Fig. 2A comparison of 23 PKS gene clusters from Calcarisporium sp. with those from other fungi and bacteria is presented ( Figure S3), while one cluster has no homologous clusters in any organisms. The PKS cluster mCaBGC21 (contig_169/39.7 kb) has 36% sequence identity with the previously uncharacterized BGC AM270194.1 c1 (localized on the contig An09c0050) from Aspergillus niger ( Figure S3). The 43.8 kb long PKS cluster mCaBGC24 (contig178) shares 46% similarity with clusters NC015711.1 c13 from Myxococcus fulvus HW-1 and NC015953.1 c16 from Streptomyces sp. SirexAA-E ( Figure S3). The PKS cluster mCaBGC25 (contig189/48.1 kb) has similarity to clusters in Streptomyces sp. and Kitasatospora setae KM-6054. The PKS cluster mCaBGC34 (contig_280/33.6 kb) has a counterpart in several fungal genomes such as Aspergillus, Metarhizium and Trichophyton ( Figure S3). The cluster mCaBGC35 (contig282/48.7 kb) shares 45% of similarity with a bacterial cluster NC_012718.1_c1 (Burkholderia glumae BGR1) and it is also found in genomes of Cordyceps militaris and Glomerella graminicola ( Figure S3). In a similar fashion, we found that mCaBGC40 (contig_358/28 kb) has homologous clusters in several strains of Streptomyces sp. ( Figure S3) Likewise, mCaBGC41 (contig_367/32.7 kb) as similarities with clusters in Metarhizium strains, but at the same time, it has 30% similarity with a cluster from Nostoc punctiforme.
Biosynthetic gene clusters in Pestalotiopsis sp. The 46 Mb assembled genome of Pestalotiopsis sp.
KF079 contains 67 BGCs, which are named mPeBGC1 to mPeBGC67 (Table S9 and Figure S4). These clusters include 22 T1 PKS clusters, 12 nonribosomal peptide synthetase (NRPS) clusters, 9 terpene clusters and one each of type III PKS (T3pks) and lantipeptide clusters. Additionally, Pestalotiopsis KF079 has six clusters of hybrid nature such as three Nrps-t1pks and one each of Hglks-t1pks, T1pks-terpene and terpene-t1pks. On top of that sixteen clusters were detected with unknown types of biosynthetic genes (Table S9). Of all these BGCs only two are represented in the MiBiG database 28 namely, mPeBGC8 and mPeBGC50. The remaining 65 BGCs are unique to Pestalotiopsis. sp., as there are no homologous clusters known or else similarity is too low. Hence, 65 out of 67 BGCs of Pestalotiopsis sp are previously uncharacterized, hence not found in MiBiG database 28 , a database of known BGCs.

PKS clusters in Pestalotiopsis sp.
The single T3pks cluster found in the genome of Pestalotiopsis sp., mPeBGC2, is localized on the scaffold00001 within a fragment of 41.4 kb, and this cluster has similarities with clusters in Cordyceps militaris and Metarhizium anisopliae ( Figure S4). There are further 22 BGCs encoding PKS gene products in the Pestalotiopsis sp. genome. Cluster mPeBGC4 is localized in the scaffold00002, spanning an about 45.8 kb fragment without significant homologies with any other known cluster ( Figure S4). Spanning about 45.6 kb on the scaffold00007, cluster mPeBGC8 is known in several ascomycetes and also in different Streptomyces strains. The cluster mPeBGC8 has no homolog, however it has very limited similarities with four known clusters (Fig. 4), namely pestheic acid biosynthetic gene cluster (BGC0000121_c1, 15% identities), monodictyphenone biosynthetic gene cluster (BGC0000101_c1, 15% identities), asperthecin biosynthetic gene cluster (BGC0000684_ c1, 10% identities), TAN-1612 biosynthetic gene cluster (BGC0000156_c1, 10% identities) and viridicatumtoxin biosynthetic gene cluster (BGC0000168_c1, 10% identities). However, due to the low similarities of 10-15%, these

Hybrid clusters in Pestalotiopsis sp.
There are three Nrps-t1pks hybrid clusters in the Pestalotiopsis sp. genome (Fig. 5B) namely mPeBGC14 (scaffold000013/57.1 kb), mPeBGC39 (scaffold000049/54.2 kb), and mPeBGC40 (scaffold000051/52.5 kb). The remaining three hybrid clusters in the Pestalotiopsis genome (Fig. 5B) are of the types T1pks-terpene, Terpene-t1pks and Hglks-t1pks respectively, and are called as mPeBGC1 (scaf-fold0000/60.4 kb), mPeBGC50 (scaffold000072/50.4 kb) and mPeBGC64 (scaffold000158/30.7 kb), respectively. None of these hybrid clusters have significant hits, even with uncharacterized clusters in other organisms ( Figure S4).  29 . We found sixteen unknown-type BGCs in Pestatiopsis genome ( Figure S4), without significant cluster homology with other micro-organisms Core PKS and NRPS proteins in the two marine fungi In the Calcarisporium KF525 genome, we identified 50 putative PKSs, including four PKS-NRPS hybrids, one PKS with an N terminal adenylation and PCP domain, six non-reducing PKSs and thirty-seven reducing PKSs (Fig. 2). The non-reducing PKS g3018 was found to be a possible orthologue of the proposed bikaverin synthase in Fusarium avenaceum (FAVG1_10226) 30 . Analyses of the remaining PKSs did not result in identification of orthologues with known products in other species.
Several bioactive compounds are identified from Calcarisporium sp. KF525 31,32 and the most fascinating one is calcaride A, which has antibacterial properties. Calcaride A belongs to the group of macrocyclic polyesters, for which the biosynthetic routes are yet to be identified. However based on aromatic ring formation, calcaride A can be produced by two possible genes namely NR-PKS (g1826) and the HR-PKS (g1824). Nevertheless, these two genes are not expressed under laboratory conditions (Fig. 2).
In Pestalotiopsis sp., putative 36 PKS were identified including four PKS-NRPS hybrides, two PKSs with N terminal adenylation and PCP domains, eight non-reducing PKSs and twenty-three reducing PKSs (Fig. 7). Twelve PKSs have an orthologue in the previously sequenced P. fici 33 including an ortholog of the P. fici pestheic acid synthase (PtaA 34 ). The non-reducing PKS g18514 shares 81% homology to the melanin polyketide synthase from Nodulisporium sp. ATCC74245 (AAD38786.1) and an orthologue in Pestalotiopsis microspore has previously been shown to be required for conidial pigmentation 35 . Another likely PKS for pigmentation was also identified in g7641, which shares 60-62% identity with fusarubin synthase (PKS3) from members of the Fusarium genus 36 . The non-reducing PKS g18600 shares 42% identity to AN6448 from Aspergillus nidulans, which produce 3-methylorsellinic acid 37 and it seems therefore likely that Pestalotiopsis sp. can produce a similar compound.
The genomes of both strains contain less NRPSs than PKSs, as we identified 32 and 18 putative NRPSs in Calcarisporium sp. and in Pestalotiopsis sp. (Fig. 8), respectively. We identified a putative iron-chelating siderophore synthetase of the ferricrocin type 38 in both species -Calcarisporium sp. (g5520) and Pestalotiopsis sp. An orthologue of the peramine synthase from Epichloe typhina was identified in Calcarisporium sp. (g14322). Peramine is a potent insect feeding deterrent synthesized by a two-module NRPS in Epichloë and has been suggested to be produced in a mutualistic interaction with its host plant perennial ryegrass in order to confer protection to against insect herbivory 39 .
Calcarisporium sp. has a MAT1-2 locus while Pestalotiopsis sp. has no detectable mating locus. The genomic fragments that carry the mating type genes are usually designated as the mating-type (MAT) locus, which regulates sexual reproduction in different fungi 40 . In self-sterile (heterothallic) species, mating occurs between morphologically identical partners that are only distinguished by their MAT locus. In filamentous ascomycetes, the MAT locus consists of two different DNA segments in the mating partners termed the MAT1-1 and MAT1-2 idiomorphs 41 . In contrast to heterothallic species, the genome of self-fertile (homothallic) filamentous ascomycetes contains genes indicative of both mating types that can be either linked or unlinked 42,43 . During genome annotation process, detection of these loci and flanking gene identification is a standard process, which aids in determination of the mode of sexual reproduction in different fungi. So far little is known about the sexual life style of marine fungi. To unravel sexual lifes of these fungi, we have performed specialized homology detections of MAT genes and corresponding loci (explained in details in Suppl. section S1). A tBLASTN search with the MAT1-2-1 HMG domain mating type protein of S. macrospora and N. crassa revealed the presence of  Figures S5 and S6. The APN2 gene encoding a putative DNA lyase and the SLA2 gene encoding a cytoskeleton assembly control factor have been reported neighboring MAT loci in many other ascomycetes 44 . Both genes, APN2 (g12882.t1) and SLA2 (g12886.t1) can be identified on the same contig_519 as the putative MAT1-2-1 gene (Fig. 9). Two further open reading frames are adjacent to MAT1-2-1 and flanked by APN2 and SLA2, g12884.t1 and g12885.t1 on contig519 (Fig. 9), which encode proteins of 242 and 133 amino acids, respectively. Transcriptomic data revealed that both mating types genes are not expressed in wild type condition, while flanking genes -APN2 (g12882.t1) and SLA2 (g12886.t1) were expressed (Fig. 9), in at low level within tiers 9 and 8 (Table 2), respectively.
Taken together, the genome data showed that the sequenced isolate Calcarisporium sp. has a MAT1-2 mating type. MAT1-1 sequences could not be identified suggesting that Calcarisporium sp. is heterothallic, providing that Calcarisporium spec is able to reproduce sexually. A novel MAT1-2 specific ORF could be identified which is related to the MAT-1-2-3 protein encoded in the MAT1-2 loci members of the Hypocreales 45 . We performed similar homology-based detection of mating type genes in Pestalotiopsis sp., which yielded no results, suggesting that the isolated Pestalotiopsis strain has no mating type genes. We have recently reported that another marine fungus S. brevicaulis LF580 was a MAT1-1 strain 15 . These finding corroborates that reproduction in marine environments can be varied nature like either vegetative and sexually or both.
Summary of carbohydrate active enzyme-encoding genes identified in the two novel marine fungal genomes. Little is known about the lifestyle and ecological importance of marine fungi. Many water environments are low in nutrients and therefore unlikely to favor organisms that feed primarily by attachment to larger physical substrates and osmotrophy 2 . Fixed carbon on land is largely invested in the construction of large and complex plant tissues rich in energy and nutrients. Digestion of these plant tissues requires a complex set of carbohydrate active enzymes. Calcarisporium and Pestalotiopsis strains used in this study were collected in the German Wadden Sea, which is an area of about 3475 square miles with one of the highest biological primary productions in the world. As such the analysis of carbohydrate active enzymes is highly relevant. Using annotation tools derived from the CAZy database (http://www.cazy.org/), we identified 949 and 476 CAZy genes in Pestalotiopsis sp. KF079 and Calcarisporium sp. KF525 genomes ( Fig. 10 and Table S10), respectively, confirming that Pestalotiopsis species are very rich in CAZymes and that Calcarisporium sp. is rather close in number to the marine derived-fungus Scopulariopsis brevicaulis LF580 46 . For Pestalotiopsis sp., the proteins encoded by the corresponding genome are divided into six major classes, namely 423 glycoside hydrolases (GH), 122 glycosyltransferases (GT), 35 polysaccharide lyases (PL), 80 carbohydrate esterases (CE), 134 carbohydrate binding module (CBM) and 155 auxiliary activities (AA). In comparison, CAZymes candidates of Calcarisporium sp. are radically lower in number for each class except for GT. Comparing the various genomes from mainly plant pathogens, entomopathogens and other model fungi, we can observe that the number of GTs, enzymes involved in the biosynthesis of oligo-and polysaccharides, is rather stable across genomes while the classes involved in the degradation processes (GH, PL, CE, CBM and AA) are highly variable and depend on the lifestyle of the fungus. The total number of CAZymes is generally high for plant pathogens and saprophytes, with the exception of Trichoderma reesei, known to produce a very efficient enzyme cocktail but poorly diversified. Inversely, the entomopathogenic fungi, Metarhizium anisopliae and Metarhizium acridum, are rather poor in all these enzyme  (Table S11). In Calcarisporium sp. on the other hand, the corresponding gene number (367) is rather close to the marine-derived S. brevicaulis strain LF580 (328 genes 15 ). Comparing the distribution of the transporters into the categories defined by the Transporter Classification Database (TCDB; [2]), an overall similar distribution of the transporters of Pestalotiopsis sp., S. brevicaulis and N. crassa could be observed, while the distribution of Calcarisporium sp. transporters is slightly different (Fig. 11). We have provided complete details of transporter genes in the supplementary section S3.

Discussion
The oceans harbour enormous biochemical diversity in terms of natural products from various organisms. Marine fungi are potent producers of natural products however they have been under the least scientific focus. To bridge this gap, we used two marine fungal strains from the German Wadden Sea (Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079) and performed genome wide scanning for the secondary metabolite genes and gene cluster (BGCs). We presented here genome sequences of two marine fungi namely Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079 and the genome sizes of these two fungi are 35 Mb and ~46 Mb, respectively. Both genomes have a low content of total repeats (1%). Three other known marine fungal genomes, namely S. brevicaulis LF580 15 , C. malorum Mo12 21 and Cryptococcus sp. Mo29 21 have comparable repeat contents, which are close to 1%. This resides within the typical 1-4% of transposable elements in fungal genomes 20 . Exceptionally, only a few fungi have higher repeat contents like in a pezizomycete, Tuber melanosporum genome 47 and several fungal genome of dothiodeomycetes 48 . However, these fungi typically have large expansions of the genome size like 125 Mb for T. melanosporum 47 .
Biosynthetic genes involved in the biosynthesis of a particular metabolite are organized in clusters and are often co-regulated and these clusters are known as biosynthetic gene clusters (BGCs) 19,49 . Exceptionally, more than one BGCs are involved in production of secondary metabolites like biosynthersis of meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans 19 . These clusters have one or two core genes and several accessary genes, which encode proteins required for biosynthesis of the metabolite. Often filamentous fungi have 25 BCGs but this number can be much higher (80-90) 50 . The two marine fungi analysed here possess a total of 127 BGCs. Most of them have no significant homologies to BGCs in other fungi as well as in the bacteria. Notably, only three of these gene clusters are well characterized BGCs (Fig. 4) and reported in MiBiG datasets 28 . Our genome analyses thus revealed a huge amount of novel BGCs, highlighting that marine fungi are an exceptional source for secondary metabolites. Future studies should be aimed at identifying conditions in which the BGCs are active or methods to activate them artificially so that their properties can be analysed in more detail. In the draft fungal genomes, genomic fragments are smaller and hence in a few cases are noted that possesses a single gene in the cluster like mCaBGC60 ( Figure S3). The cluster mCaBGC60 is localized on the contig1101, which is only 11.9 kb    in size. Hence, drawing a conclusion from this cluster will be difficult, yet kept as found, and further genome assembly improvements and/or resequencing can resolve issues of such clusters.
Pestalotiopsis and Calcarisporium species belong to the Xylariales and Hypocreales, respectively, and are distributed in tropical and temperate regions (for a review on Pestalotiopsis see 51 ). Pestalotiopsis species typically are phytopathogens causing different diseases i.e. needle or tip blights, canker lesions or fruit rots. Pestalotiopsis sp. are also found as endophytes or saprophytes, as they were isolated from dead leaves, barks and twigs. In addition, some species were found in animal infections demonstrating that these fungi could have versatile nutritional habits, or are opportunistic. In general, Pestalotiopsis sp. appears to be host-specific and live in a wide range of substrates or associated with different hosts. Calcarisporium sp. have a widespread occurrence and they are generally isolated as endophytes or parasites from basidiomycetes 52 , or ascomycetes, or from wood 53 , but have occasionally been isolated from marine environments 32 . In a recent study of a mangrove fungus, Pestalotiopsis sp. was described along with its adaptation to sea salt 54 . By a proteomic analysis, it was demonstrated that the lignocellulolytic enzyme composition was considerably changed in salt conditions, with for instance, a great reduction of the oxidase abundance, while specific carbohydrate-active enzymes are secreted exclusively at high salt concentrations. Taking into account all these data related to the CAZyome and sugar transporter repertoires (Tables S10-S11), it can be suggested that Pestalotiopsis sp. KF079, similar to the marine-derived S. brevicaulis, possesses a metabolism focused on the breakdown of the terrestrial plant biomass rather than algal or animal biomass. On the other hand, Calcarisporium sp. has adopted a different (and reduced) CAZy repertoire specialized for algae and animal degradation that reflects a life style oriented either towards parasitism or endophytic growth or towards utilization of dead algae or animals.
Upon scanning MAT loci in these two marine fungi, we demonstrated that Calcarisporium sp. has a MAT1-2 mating type while no MAT locus was detected in Pestalotiopsis sp.
Altogether, we present two draft genomes of marine fungi with a high number of BGC most of them being new clusters not observed before. These computational predictions of the genomic data provide further insight into genetics of these two fungi. This study step platform for the designing experiments to confirm findings about BGCs and corresponding secondary metabolite biosynthesis. We provide a comprehensive annotation of these genomes.

Methods
Fungal strain collection, cultivation, and DNA isolation. These two marine fungal strains (Pestalotiopsis sp. KF079 and Calcarisporium sp. KF525) were isolated from the German Wadden Sea, which is the southeastern part of the North Sea. These strains were cultivated as previously described 55 . DNA isolation was per formed as described in the supplementary section S4. Unraveling and characterization of biosynthetic gene clusters. Initially, putative genes that encoding for proteins which produce bioactive compounds are identified using BLAST 58 with an E-value < 1e −3 . Subsequently, this genome was analyzed using antiSMASH 3.0 29 for putative clusters and further examined by manually coupled with RNA-Seq data. The functional domains of PKSs and NRPSs were identified as previously described 59 , using a combinations of tools namely antiSMASH 3.0 29 , NCBI Conserved Domain Database 60 , InterPro 24 and the PKS/NRPS Analysis Web-site 61 .
Data access. Entire datasets used in the current work were publically available using BioSample accession IDs: SAMN06272793 and SAMN06272794 with corresponding BioProject accession IDs as PRJNA368776 and PRJNA368777 for Pestalotiopsis sp. KF079 and Calcarisporium sp. KF525, respectively.