Advertisement

Symbiosis

, Volume 66, Issue 2, pp 99–106 | Cite as

Diversity of fungi isolated from three temperate ascidians

  • Susanna López-LegentilEmail author
  • Patrick M. Erwin
  • Marta Turon
  • Oded Yarden
Short Communication

Abstract

Ascidians are known to harbor diverse and host-specific bacterial and archaeal communities in their tunic. However, to date, only one ascidian species has been investigated to assess symbiotic relationships with fungi and the extent of their diversity. In this study, we isolated and identified 37 strains of fungi in association with three common ascidian species in the NW Mediterranean Sea: Cystodytes dellechiajei, Didemnum fulgens, and Pycnoclavella communis, and 15 additional strains from concentrated seawater samples collected around the animals. Most of the isolated fungi were classified within four orders: Eurotiales (predominantly Penicillium spp.), Pleosporales, Hypocreales (predominantly Trichoderma spp.), and Capnodiales (Cladosporium spp.). Three additional fungal isolates from C. dellechiajei and D. fulgens belonged to the orders Helotiales, Phylachorales and Microascales, and matched to well-known plant and human pathogens (Botrytis cinerea, Plectosphaerella cucumerina and Scopulariopsis brevicaulis). Host-specificity of ascidian-associated fungi was not apparent and thus the significance of ascidian-fungal associations for ascidian wellbeing and their possible ecological roles remain unknown.

Keywords

Fungus Mediterranean Sea ITS Sea-squirt Tunicata Pathogens 

1 Introduction

Invertebrate-bacterial associations and, to a lesser degree, invertebrate-archaeal associations have been widely described in the literature (e.g., Mouchka et al. 2010; Webster and Taylor 2012; Erwin et al. 2014). Most of these studies have also demonstrated a high degree of specificity between the invertebrate host and at least a core community of its symbionts (Taylor et al. 2004; Ainsworth et al. 2010, 2015; Schmitt et al. 2012; Easson and Thacker 2014; Erwin et al. 2014). Moreover, some of these associations are obligate, in that both the host and the symbiont depend on each other for their long-term survival (Ainsworth et al. 2010; Thacker and Freeman 2012). Other potential invertebrate-microbial associations include symbioses with eukaryotes (e.g., dinoflagellates in corals; reviewed in Rowan 1998) and fungi (reviewed in Yarden 2014). When compared with other reported symbiotic relationships, the latter has been surprisingly understudied and has mostly focused on corals and sponges. To date, we know that coral-associated fungi are prevalent and that in at least a few species these associations are host-specific rather than environmentally determined (e.g., Amend et al. 2012). In sponges, hundreds of fungal strains have been isolated and identified as either marine or ubiquitous taxa (Höller et al. 2000; Morrison-Gardiner 2002; Li and Wang 2009; Menezes et al. 2010; Paz et al. 2010; Wiese et al. 2011) with several fungal strains producing previously unknown secondary metabolites (Höller et al. 2000; Paz et al. 2010; Wiese et al. 2011; Koch et al. 2014). However, evidence for host-specificity is still rare, and has only been demonstrated in a few cases (Li and Wang 2009).

Ascidians or sea-squirts (Chordata, Tunicata) are conspicuous benthic sessile invertebrates found all around the world (Shenkar and Swalla 2011). Ascidians are especially abundant in hard bottom rocky substrates and attached to artificial substrates, such as docks and aquaculture cages (Lambert 2001; Carman et al. 2010). With few exceptions, ascidians are filter-feeding organisms with an abundance and diversity of species and functions that render them critical to healthy ecosystem functioning (Lambert 2005). As sessile invertebrates, ascidians are also easy prey for predators and thus have developed a diverse array of defensive secondary metabolites (Pisut and Pawlik 2002; Tarjuelo et al. 2002; López-Legentil et al. 2006b). In fact, ascidians are well-known producers of marine natural products (Paul et al. 2011; Blunt et al. 2014, 2015) and several of these secondary metabolites have also shown interesting pharmaceutical properties, especially as anti-cancer drugs (Erwin et al. 2010). Although the importance of many of these compounds is well established, to date there is little information about exactly who produces them, since ascidians are known to establish complex symbiotic associations with a wide range of bacteria and archaea (Tait et al. 2007; Erwin et al. 2013, 2014; Tianero et al. 2015). Another possible source of these bioactive compounds are fungal associates (Xin et al. 2007; Montenegro et al. 2012), but this potentially important symbiotic association between ascidians and fungi has been largely understudied. To the best of our knowledge, only one previous study has described the diversity of culturable fungi associated with an ascidian and has found that the colonial species Didemnum sp. (sampled in Brazil) hosted a higher diversity of filamentous fungi than the investigated, sympatric sponge species (i.e., Mycale laxissima, Amphimedon viridis and Dragmacidon reticulata; Menezes et al. 2010).

In this study, we targeted three colonial ascidians commonly found in rocky bottom habitats of the NW Mediterranean Sea: Cystodytes dellechiajei, Didemnum fulgens, and Pycnoclavella communis. The species C. dellechiajei is distributed worldwide in tropical and temperate waters and exhibits high phenotypic plasticity with many color morphs and chemotypes (López-Legentil et al. 2005; López-Legentil and Turon 2005). The purple morph in particular, is one of the most abundant morphotypes in the NW Mediterranean Sea and produces a set of secondary metabolites classified as pyridoacridine alkaloids (López-Legentil et al. 2005; Bontemps et al. 2010; Bry et al. 2011). Several of these metabolites are known to have anti-predatory (López-Legentil et al. 2006b), antimicrobial (Bontemps et al. 2010) and anti-tumoral (Martínez-García et al. 2007a) activities. In addition, the production of the four most common pyridoacridine alkaloids (namely shermilamine B, kuanoniamine D, and their deacetylated forms) in the purple morph were quantified over time and shown to lack statistically significant variation, although minimum values were consistently recorded in late summer after the reproductive period of the species (López-Legentil et al. 2006a). Finally, C. dellechiajei is also known to host a wide array of bacterial symbionts (Martínez-García et al. 2007b), including oxygenic phototrophs containing chlorophylls a, b, c, and d (Martínez-García et al. 2011).

D. fulgens exhibits a patchy distribution along the NE coast of the Iberian Peninsula, reaching densities of >12 colonies per meter square in some areas (López-Legentil et al. 2013). This species is also known to harbor a stable bacterial community in its tunic with at least some of these symbionts being transferred to the larvae (López-Legentil et al. 2015). D. fulgens chemistry has not yet been investigated, however this species is likely to produce bioactive secondary metabolites given the prevalence of bioactive species in this ascidian genus (Blunt et al. 2015; and earlier reviews). Finally, P. communis is one of the most common colonial ascidians reported along the western Mediterranean shores (Pérez-Portela et al. 2007). Related species in the genus Pycnoclavella are known to produce bioactive secondary metabolites (Appleton et al. 2002; Appleton and Copp 2003) and P. communis in particular appears to be chemically defended against predators (Pérez-Portela and Turon 2007).

The goal of this study was to determine whether culturable fungi could be successfully isolated from three common ascidian species in the NW Mediterranean Sea, and if so, assess their diversity by DNA sequence analysis of the nuclear ribosomal internal transcribed spacer (ITS) region. To achieve these objectives, fungi were isolated and identified from three ascidian species (C. dellechiajei, D. fulgens, and P. communis) collected at three time points (May 2012, Oct 2012, May 2013) and from triplicate ambient seawater samples (collected May 2013).

2 Material and methods

2.1 Sample collection

Samples were collected from L’Escala, Spain (‘La Depuradora’: 42° 7′ 29″ N, 3° 7′ 57″ E; NW Mediterranean Sea) at depths <12 m during three collection trips carried out on May 18, 2012 (seawater temperature 16 °C), October 4, 2012 (seawater temperature 18 °C) and May 10, 2013 (seawater temperature 15 °C). Three species of colonial ascidians were targeted: the purple morph of Cystodytes dellechiajei (Della Valle, 1877), Didemnum fulgens (Milne-Edwards, 1841), and Pycnoclavella communis Pérez-Portela, Duran and Turon, 2007 (Fig. 1). Ascidian and seawater samples (1L) were collected by SCUBA, brought to the surface and immediately processed. The exterior of each ascidian species was rinsed and gently dried to remove loosely attached surface organisms, then placed into a sterile petri dish for dissection on site. For C. dellechiajei and D. fulgens, each colony was dissected in 2 to 3 thin bands (1 to 2 mm wide, 2 to 3 cm in length, < 0.5 cm in height) that included both tunic tissue and zooids. In addition, three zooids of C. dellechiajei were isolated from the tunic and plated separately. For P. communis, each colony was dissected into 6 zooid plus tunic sections. Different dissection methods were employed to accommodate differences in zooid size (>1 mm in C. dellechiajei, <1 mm in D. fulgens, 2–3 mm in P. communis) and arrangement (C. dellechiajei and D. fulgens zooids are embedded in a common tunic, while P. communis zooids are connected by tunic strings and tightly surrounded by individual tunics). All ascidian dissections were immediately placed on culture plates (see below) and into an insulated cooler for transport to the laboratory (ca. 2 h transit time). On the final collection trip (May 10, 2013), we also collected triplicate ambient seawater samples (1 L each). Seawater samples were collected in close proximity of the ascidians (<4 m) in 3 sterile Nalgene bottles (one per replicate), concentrated on 0.2 μm polycarbonate filters (Whatman) and placed upside down (i.e., filtrate in direct contact with culture media) on 3 separate culture plates (see below).
Fig. 1

In situ photographs of the colonial ascidians Cystodytes dellechiajei (a), Didemnum fulgens (b), and Pycnoclavella communis (c; indicated by arrows) in L’Escala, Spain (NW Mediterranean Sea). Scale bars: 2 cm. Insets show representative fungi growing from tunic slices of C. dellechiajei (a) and D. fulgens (b), and the whole zooid (including the tunic) of P. communis (c; zooids are buried under the growing fungi). Scale bar: 1 cm

2.2 Fungal isolation

Tunic bands and zooids dissected from the different ascidians and seawater filtrates were used to inoculate culture plates containing malt extract agar (Merckoplate®, Merck, Kenilworth, NJ, USA) to grow a wide spectrum of fungi and amended with the antibacterial chemicals chloramphenicol and gentamycin. Plates were incubated in the dark (to mimic conditions in the tunic) for 6 days at room temperature. Once growth was visible, different fungal morphotypes (based on color and shape differences) were isolated and streaked to purity on separate culture plates. Fungi were then allowed to grow for 7 days before a sample of each pure culture was collected for DNA extraction.

2.3 DNA extraction and ITS amplification

DNA was extracted using the Animal Tissue Protocol, DNeasy® Blood and Tissue kit (Qiagen®) and used as template for PCR amplification of fungal internal transcribed spacer (ITS) gene sequences using the forward primer ITS1 and the reverse primer ITS4 (White et al. 1990). The thermocycler program consisted of an initial denaturing step at 94 °C for 6 min, 35 amplification cycles (denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 40 s), and a final extension at 72 °C for 7 min, performed on a PCR System 9700 (Applied Biosystems). Cleaning and sequencing reactions were carried out by Macrogen, Inc. (Seoul, Korea) and consensus sequences (see below) were deposited in the GenBank database (Table 1).
Table 1

Fungal taxa isolated from the colonial ascidians Cystodytes dellechiajei, Didemnum fulgens, and Pycnoclavella communis and from ambient seawater samples

Ascidian species

Col. date

Fungal order

Potential species

Code

Acc. no.

Cystodytes dellechiajei

18-May-12

Eurotiales

Penicillium sp.

CD-1

KT121502

18-May-12

Eurotiales

Aspergillus sp.

CD-3

KT121532

4-Oct-12

Pleosporales

Epicoccum nigrum

CD-1b

KT121518

4-Oct-12

Pleosporales

Alternaria sp.

CD-2b

KT121535

4-Oct-12

Pleosporales

Phoma sp.

CD-3b

KT121495

4-Oct-12

Eurotiales

Penicillium sp.

CD-4b

KT121501

10-May-13

Phylachrocales

Plectosphaerella sp.

1CD-1

KT121494

10-May-13

Capnodiales

Cladosporium sp.

1CD-2

KT121528

10-May-13

Hypocreales

Fusarium sp.

1CD-3

KT121517

10-May-13

Eurotiales

Penicillium brevicompactum

2CD-1

KT121512

10-May-13

Eurotiales

Penicillium sp.

3CD-1

KT121509

10-May-13

Helotiales

Botrytis cinerea

3CD-2

KT121529

Didemnum fulgens

18-May-12

Eurotiales

Penicillium sp.

DF-2

KT121500

18-May-12

Capnodiales

Cladosporium sp.

DF-3

KT121520

18-May-12

Eurotiales

Penicillium sp.

DF-4

KT121499

4-Oct-12

Botryosphaeriales

Microdiplodia sp.

DF-1B

KT121516

10-May-13

Microascales

Scopulariopsis sp.

1DF-1

KT121492

10-May-13

Eurotiales

Penicillium brevicompactum

1DF-2

KT121515

10-May-13

Capnodiales

Cladosporium sp.

1DF-3

KT121527

10-May-13

Eurotiales

Penicillium brevicompactum

1DF-4

KT121514

10-May-13

Hypocreales

Trichoderma sp.

2DF-1

KT121488

10-May-13

Eurotiales

Penicillium brevicompactum

2DF-2

KT121511

10-May-13

Hypocreales

Clonostachys sp.

2DF-3

KT121519

10-May-13

Eurotiales

Penicillum sp.

3DF-1

KT121508

10-May-13

Capnodiales

Cladosporium sp.

3DF-2

KT121522

10-May-13

Eurotiales

Penicillium rubens

3DF-3

KT121507

10-May-13

Hypocreales

Trichoderma harzianum

3DF-4

KT121486

Pycnoclavella communis

4-Oct-12

Eurotiales

Penicillium sp.

PN-1b

KT121498

4-Oct-12

Eurotiales

Penicillium sp.

PN-2b

KT121497

4-Oct-12

Hypocreales

Bionectria sp.

PN-3b

KT121530

4-Oct-12

Eurotiales

Talaromyces sp.

PN-4b

KT121491

4-Oct-12

Eurotiales

Aspergillus fumigatus

PN-5b

KT121531

10-May-13

Hypocreales

Trichoderma harzianum

1PN-1

KT121490

10-May-13

Hypocreales

Trichoderma sp.

1PN-2

KT121489

10-May-13

Capnodiales

Cladosporium sp.

2PN-1

KT121526

10-May-13

Capnodiales

Cladosporium sp.

2PN-2

KT121525

10-May-13

Pleosporales

Phoma sp.

3PN-1

KT121496

Seawater samples

10-May-13

Eurotiales

Aspergillus sp.

1SW-1

KT121534

10-May-13

Eurotiales

Penicillium rubens

1SW-2

KT121513

10-May-13

Pleosporales

Pleosporales sp.

1SW-4

KT121493

10-May-13

Pleosporales

Alternaria sp.

1SW-5

KT121537

10-May-13

Eurotiales

Aspergillus sp.

1SW-6

KT121533

10-May-13

Hypocreales

Trichoderma sp.

2SW-1

KT121487

10-May-13

Pleosporales

Alternaria sp.

2SW-2

KT121536

10-May-13

Capnodiales

Cladosporium sp.

2SW-3

KT121524

10-May-13

Eurotiales

Penicillium chrysogenum

2SW-4

KT121523

10-May-13

Capnodiales

Cladosporium sp.

2SW-5

KT121503

10-May-13

Eurotiales

Penicillium rubens

3SW-1

KT121506

10-May-13

Capnodiales

Cladosporium sp.

3SW-2

KT121521

10-May-13

Eurotiales

Penicillium rubens

3SW-3

KT121505

10-May-13

Eurotiales

Penicillium rubens

3SW-4

KT121504

10-May-13

Eurotiales

Penicillium rubens

3SW-5

KT121503

‘Col. Date’ indicates when the samples were collected (day, month and year). ‘Code’ corresponds to the unique code given to each isolate obtained in this study. ‘Acc. No.’ refers to the GenBank accession number of each isolate’s ITS sequence

2.4 DNA sequence and phylogenetic analyses

Raw sequence reads were processed in Geneious v. 8 (Kearse et al. 2012) by aligning forward and reverse reads to yield final consensus sequences. Each consensus sequence was then analyzed using the BLASTn tool from GenBank to assess taxonomic affiliations. For phylogenetic analyses, all ascidian-derived sequences were aligned using Clustal W v. 2 (Larkin et al. 2007) with a gap opening penalty of 24 and a gap extension penalty of 4. A phylogenetic tree was built using the neighbor-joining (NJ) method implemented in the Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0 (Tamura et al. 2013). Data were re-sampled using 1000 bootstrap replicates (Felsenstein 1985).

3 Results and discussion

A total of 52 different fungal strains were successfully isolated from all ascidian and seawater samples, except for Pycnoclavella communis collected on May 18, 2012 and all zooid dissections of Cystodytes dellechiajei. The absence of fungal isolates from C. dellechiajei zooids (where active filtration of seawater occurs) suggests that most fungi are located in the tunic of the animal and that the tunic represents a more suitable habitat for the survival or long-term storage of fungal spores. Moreover, fungi were observed growing out from the cut edges of the tunic (Fig. 1a), indicating that fungi are located within the tunic and do not just adhere to the surface exposed to the environment. Similarly, fungi isolated from Didemnum fulgens appeared to sprout from within the tunic (Fig. 1b), although in this case and also for Pycnoclavella communis (Fig. 1c), we were unable to separate zooids from their surrounding tunic and cannot determine exactly where fungal growth originated. Together, these observations suggest that at least some fungal propagules are not expelled or rapidly digested by the zooid, but rather reside (or are stored) in the tunic where they remain in a viable state for some time.

Of the 52 sequences obtained here, 37 sequences were retrieved from fungal isolates associated with ascidians and 15 from seawater samples. Ascidian-associated fungi were classified in eight distinct orders: Eurotiales, Capnodiales, Helotiales, Microascales, Phylachorales, Hypocreales, Botryosphaeriales and Pleosporales (Table 1). A high degree of host-specificity of ascidian-associated fungi was not apparent, since different individuals within the same host species appeared to contain different fungal isolates (Table 1). Differences among replicates could be due to a number of reasons, including different sampling times (and thus abiotic conditions) and intraspecific variability. Despite such variability, most of the fungal sequences retrieved for all host species and replicates were classified within the four fungal orders: Eurotiales, Pleosporales, Hypocreales, and Capnodiales (Table 1, Fig. 2). Moreover, all sequences obtained for P. communis and the seawater samples were classified within the above mentioned four orders (Table 1, Fig. 2). Similarly, Menezes et al. (2010) found that the most common fungi in the Brazilian ascidian Didemnum sp. (>10 % of all isolates) belonged to three of the major orders reported here (Eurotiales, Pleosporales, and Hypocreales). Interestingly, these orders have also been commonly reported fungal orders in the Mediterranean sponge Ircinia variabilis (Paz et al. 2010) and in the Caribbean coral Acropora formosa (Yarden et al. 2007), and contain sequences of both marine and terrestrial origin.
Fig. 2

Neighbor-joining phylogenetic tree based on rRNA ITS sequences of cultured fungi associated with the Mediterranean ascidians Cystodytes dellechiajei (CD), Didemnum fulgens (DF), and Pycnoclavella communis (PC). Fungal order and the number of strains (in parenthesis) are also shown. Numbers above or below branches correspond to bootstrap values

Terrestrial fungal strains are often retrieved from marine invertebrates (e.g., Li and Wang 2009; Menezes et al. 2010; Paz et al. 2010) and, in most cases, are believed to represent spore contaminants, not true symbionts (reviewed in Suryanarayanan 2012). For instance, Penicillum rubens has been commonly retrieved from several macro-organisms, sand and seawater samples (Park et al. 2014) and was isolated from both D. fulgens and seawater samples herein. Similarly, Aspergillus fumigatus (order Eurotiales) is ubiquitous on land and has also been observed in association with diverse marine invertebrates (e.g., sea cucumbers, soft corals and P. communis herein). In contrast, some marine-derived fungi appear to exhibit host-specificity and be involved in host metabolism to some degree (e.g., producing or co-producing bioactive secondary metabolites). For example, Epicoccum nigrum (order Pleosporales) has been isolated from sponges, produces cytotoxic metabolites (Jiao et al. 2009; Sun et al. 2011), and here it was only observed in C. dellechiajei. These observations indicate some overlapping specificity patterns among fungi isolated from ascidians and other marine invertebrates, yet there remains little data regarding the host-specificity and ecological roles of ascidian-associated fungi.

Some fungal isolates obtained from the ascidians C. dellechiajei and D. fulgens belonged to non-commonly retrieved orders (Fig. 2) and matched to well-known plant pathogens. C. dellechiajei isolate 3CD-2 (Table 1) was classified within the order Helotiales and matched to Botrytis cinerea (100 % identity; 100 % coverage), a necrotrophic fungus responsible for gray mold on hundreds of dicot plants (Elad et al. 2007; Williamson et al. 2007). A second isolate from this ascidian species (1CD-1; Table 1) belonged to the order Phylachorales and matched to Plectosphaerella cucumerina (100 % identity; 100 % coverage), another well-known pathogen of several plant species (Carlucci et al. 2012) that has also been reported in marine sediments and that produces several bioactive secondary metabolites (Carr et al. 2009). Finally, isolate 1DF-1 from D. fulgens was classified within the order Microascales and was closely related to Scopulariopsis brevicaulis (99 % identity; 100 % coverage), a human pathogen that can cause severe illnesses (e.g., skin lesions, pneumonia; Wheat et al. 1984; Dhar and Carey 1993; Cuenca-Estrella et al. 2003). More recently, S. brevicaulis strain LF580 was isolated from a Mediterranean sponge and was shown to produce scopularides A and B (Yu et al. 2008), both exhibiting specific activities against pancreatic and colon tumor cells (Imhoff et al. 2010). The role of some marine invertebrates as potential pathogens reservoirs has been previously suggested. For instance, the fungus Aspergillus sydowii can cause aspergillosis in Caribbean sea fans (Geiser et al. 1998; Alker et al. 2001) and a coral-infectious isolate has been isolated from an apparently healthy Bahamian sponge (Ein-Gil et al. 2009). The finding of putative pathogenic fungi in ascidians indicates another possible reservoir of disease-causing propagules in marine invertebrates, although their relative importance remains largely unknown. Indeed, the transmission of a potential pathogen from its marine animal reservoir to its target has never been reported and it is not yet clear how a fungus may escape the inner tissues of an ascidian or a sponge and be released to the environment, to then become an active pathogenic agent. While the potential impact of zoonotic diseases on marine mammals has been drawing attention (including the threat posed by fungal pathogens; reviewed in Van Bressem et al. 2009), and in spite of a growing awareness to marine invertebrate diseases (e.g., Webster 2007; Bourne et al. 2009) much less attention has been given to the potential zoonotic nature of some of these diseases.

Ascidians, like sponges, are active filter feeders and thus constantly filter huge volumes of seawater and remove microbes of all types from the water column. Depending on their size and nature, these microbes may be expelled from the ascidian, digested as prey items or remain undigested and accumulate in host tissues. Microbes present in the ascidian tunic or the sponge mesohyl may remain viable for a period of time, and in the case of bacteria, are able to reproduce and maintain stable communities for long periods of time (Erwin et al. 2012; López-Legentil et al. 2015). Fungi in the ascidian tunic are probably stored as spores, since fungal hyphae (easier to identify in light and electron micrographs than spores) have never been reported. The ecological role of these spores and the potential contribution of fungi to host metabolism and overall fitness remain unclear. However, evidence to date has shown a surprising diversity of fungi in ascidians (Menezes et al. 2010; this study), warranting further investigations into the significance and prevalence of this symbiotic relationship and for bioprospecting. In particular, future metagenomic and transcriptomic studies should provide a more comprehensive data set to assess which fungal taxa are establishing true symbiotic relationships with their host (if any) and their possible involvement in the production of bioactive secondary metabolites.

Notes

Acknowledgments

This research was funded by the Spanish Government project MARSYMBIOMICS CTM2013-43287-P, and the Catalan Government grant 2014SGR-336 for Consolidated Research Groups.

References

  1. Ainsworth TD, Thurber RV, Gates RD (2010) The future of coral reefs: a microbial perspective. Trends Ecol Evol 25:233–240CrossRefPubMedGoogle Scholar
  2. Ainsworth TD, Krause L, Bridge T, Torda G, Raina J-B, Zakrzewski M, Gates RD, Padilla-Gamino JL, Spalding HL, Smith C, Woolsey ES, Bourne DG, Bongaerts P, Hoegh-Guldberg O, Leggat W (2015) The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts. ISME J in pressGoogle Scholar
  3. Alker AP, Smith GW, Kim K (2001) Characterization of Aspergillus sydowii (Thom et Church), a fungal pathogen of Caribbean sea fan corals. Hydrobiologia 460:105–111CrossRefGoogle Scholar
  4. Amend AS, Barshis DJ, Oliver TA (2012) Coral-associated marine fungi form novel lineages and heterogenous assemblages. ISME J 6:1291–1301PubMedCentralCrossRefPubMedGoogle Scholar
  5. Appleton DR, Copp BR (2003) Kottamide E, the first example of a natural product bearing the amino acid 4-amino-1, 2-dithiolane-4-carboxylic acid (Adt). Tetrahedron Lett 44:8963–8965CrossRefGoogle Scholar
  6. Appleton DR, Page MJ, Lambert G, Berridge MV, Copp BR (2002) Kottamides A–D: novel bioactive imidazolone-containing alkaloids from the New Zea- land ascidian Pycnoclavella kottae. J Org Chem 67:5402–5404CrossRefPubMedGoogle Scholar
  7. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR (2014) Marine natural products. Nat Prod Rep 31:160–258CrossRefPubMedGoogle Scholar
  8. Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR (2015) Marine natural products. Nat Prod Rep 32:116–211CrossRefPubMedGoogle Scholar
  9. Bontemps N, Bry D, López-Legentil S, Simon-Levert A, Long C, Banaigs B (2010) Structures and antimicrobial activities of pyridoacridine alkaloids isolated from different chromotypes of the ascidian Cystodytes dellechiajei. J Nat Prod 73:1044–1048CrossRefPubMedGoogle Scholar
  10. Bourne DG, Garren M, Work TM, Rosenberg E, Smith GW, Harvell CD (2009) Microbial disease and the coral holobiont. Trends Microbiol 17:554–562CrossRefPubMedGoogle Scholar
  11. Bry D, Banaigs B, Long C, Bontemps N (2011) New pyridoacridine alkaloids from the purple morph of the ascidian Cystodytes dellechiajei. Tetrahedron Lett 52:3041–3044CrossRefGoogle Scholar
  12. Carlucci A, Raimondo ML, Santos JC, Phillips AJL (2012) Plectosphaerella species associated with root and collar rots of horticultural crops in southern Italy. Persoonia 28:34–48PubMedCentralCrossRefPubMedGoogle Scholar
  13. Carman MR, Morris JA, Karney RC, Grunden DW (2010) An initial assessment of native and invasive tunicates in shellfish aquaculture of the North American east coast. J Appl Ichthyol 26:8–11CrossRefGoogle Scholar
  14. Carr G, Tay W, Bottriell H, Andersen SK, Mauk AG, Andersen RJ (2009) Plectosphaeroic acids A, B, and C, Indoleamine 2,3-Dioxygenase inhibitors produced in culture by a marine isolate of the fungus Plectosphaerella cucumerina. Org Lett 11:2996–2999CrossRefPubMedGoogle Scholar
  15. Cuenca-Estrella M, Gomez-Lopez A, Mellado E, Buitrago MJ, Monzón A, Rodriguez-Tudela JL (2003) Scopulariopsis brevicaulis, a fungal pathogen resistant to broad-spectrum antifungal agents. Antimicrob Agents Chemother 47:2339–2341PubMedCentralCrossRefPubMedGoogle Scholar
  16. Dhar J, Carey PB (1993) Scopulariopsis brevicaulis skin lesions in an AIDS patient. AIDS 7:1283–1284CrossRefPubMedGoogle Scholar
  17. Easson CG, Thacker RW (2014) Phylogenetic signal in the community structure of host-specific microbiomes of tropical marine sponges. Front Microbiol 5:Article 532CrossRefPubMedGoogle Scholar
  18. Ein-Gil N, Ilan M, Carmeli S, Smith GW, Pawlik JR, Yarden O (2009) Presence of Aspergillus sydowii, a pathogen of gorgonian sea fans in the marine sponge Spongia obscura. ISME J 3:752–755CrossRefPubMedGoogle Scholar
  19. Elad Y, Williamson B, Tudzynski P, Delen N (2007) Botrytis spp. and diseases they cause in agricultural systems—an Introduction. In: Elad Y, Williamson B, Tudzynski P, Delen N (eds) Botrytis: biology, pathology and control. Springer, The NetherlandsCrossRefGoogle Scholar
  20. Erwin PM, López-Legentil S, Schuhmann PW (2010) The pharmaceutical value of marine biodiversity for anti-cancer drug discovery. Ecol Econ 70:445–451CrossRefGoogle Scholar
  21. Erwin PM, Pita L, López-Legentil S, Turon X (2012) Stability of sponge-associated bacteria over large seasonal shifts in temperature and irradiance. Appl Environ Microbiol 78:7358–7360PubMedCentralCrossRefPubMedGoogle Scholar
  22. Erwin PM, Pineda MC, Webster N, Turon X, López-Legentil S (2013) Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata. Symbiosis 59:35–46CrossRefGoogle Scholar
  23. Erwin PM, Pineda MC, Webster NS, Turon X, López-Legentil S (2014) Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians. ISME J 8:575–588PubMedCentralCrossRefPubMedGoogle Scholar
  24. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  25. Geiser DM, Taylor JW, Ritchie KB, Smith GW (1998) Cause of sea fan death in the West Indies. Nature 394:137–138CrossRefGoogle Scholar
  26. Höller U, Wright AD, Matthée GF, Konig GM, Draeger S, Aust H-J, Schulz B (2000) Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycol Res 104:1354–1365CrossRefGoogle Scholar
  27. Imhoff JF, Kajahn I, Lang G, Wiese J, Peters A (2010) Production and use of antimumoral, antibiotic and insecticidal cyclodepsipeptides. Patent WO 2010/142258Google Scholar
  28. Jiao J-Y, Zhu T-J, Zhu W-M, Du L, Wang C-Y, Guan H-S, Gu QQ (2009) Isolation of sponge-associated fungi and screening of their antitumor activity. J Ocean Univ China 39:42–46Google Scholar
  29. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond AJ (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649PubMedCentralCrossRefPubMedGoogle Scholar
  30. Koch L, Lodin A, Herold I, Ilan M, Carmeli S, Yarden O (2014) Sensitivity of Neurospora crassa to a marine-derived Aspergillus tubingensis anhydride exhibiting antifungal activity that is mediated by the MAS1 protein. Marine Drugs 12:4713–4731PubMedCentralCrossRefPubMedGoogle Scholar
  31. Lambert G (2001) A global overview of ascidian introductions and their possible impact on the endemic fauna. In: Sawada H, Tokosawa H, Lambert CC (eds) The biology of ascidians. Springer, Tokyo, JapanGoogle Scholar
  32. Lambert G (2005) Ecology and natural history of the protochordates. Can J Zool 83:34–50CrossRefGoogle Scholar
  33. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  34. Li Q, Wang G (2009) Diversity of fungal isolates from three Hawaiian marine sponges. Microbiol Res 164:233–241CrossRefPubMedGoogle Scholar
  35. López-Legentil S, Turon X (2005) How do morphotypes and chemotypes relate to genotypes? The colonial ascidian Cystodytes (Polycitoridae). Zool Scr 34:3–14CrossRefGoogle Scholar
  36. López-Legentil S, Dieckmann R, Bontemps-Subielos N, Turon X, Banaigs B (2005) Qualitative variation of alkaloids in color morphs of Cystodytes (Ascidiacea). Biochem Syst Ecol 33:1107–1119CrossRefGoogle Scholar
  37. López-Legentil S, Bontemps-Subielos N, Turon X, Banaigs B (2006a) Temporal variation in the production of four secondary metabolites in a colonial ascidian. J Chem Ecol 32:2079–2084CrossRefPubMedGoogle Scholar
  38. López-Legentil S, Turon X, Schupp P (2006b) Chemical and physical defenses against predators in Cystodytes (Ascidiacea). J Exp Mar Biol Ecol 332:27–36CrossRefGoogle Scholar
  39. López-Legentil S, Erwin PM, Velasco M, Turon X (2013) Growing or reproducing in a temperate sea: optimization of resource allocation in a colonial ascidian. Invertebr Biol 132:69–80CrossRefGoogle Scholar
  40. López-Legentil S, Turon X, Espluga R, Erwin PM (2015) Temporal stability of bacterial symbionts in a temperate ascidian. Front Microbiol. doi: 10.3389/fmicb.2015.01022
  41. Martínez-García M, Díaz-Valdés M, Ramos-Esplà A, Salvador N, Lopez P, Larriba E, Antón J (2007a) Cytotoxicity of the ascidian Cystodytes dellechiajei against tumor cells and study of the involvement of associated microbiota in the production of cytotoxic compounds. Marine Drugs 5:52–70PubMedCentralCrossRefPubMedGoogle Scholar
  42. Martínez-García M, Díaz-Valdés M, Wanner G, Ramos-Esplà A, Antón J (2007b) Microbial community associated with the colonial ascidian Cystodytes dellechiajei. Environ Microbiol 9:521–534CrossRefPubMedGoogle Scholar
  43. Martínez-García M, Koblízek M, López-Legentil S, Antón J (2011) Epibiosis of oxygenic phototrophs containing chlorophylls a, b, c, and d on the colonial ascidian Cystodytes dellechiajei. Microb Ecol 61:13–19CrossRefPubMedGoogle Scholar
  44. Menezes CB, Bonugli-Santosa RC, Miquelettoa PB, Passarinia MR, Silvaa CH, Justoa MR, Leala RR, Fantinatti-Garbogginia F, Oliveiraa VM, Berlinckb RG, Sette LD (2010) Microbial diversity associated with algae, ascidians and sponges from the north coast of São Paulo state, Brazil. Microbiol Res 165:466–482CrossRefPubMedGoogle Scholar
  45. Montenegro TGC, Rodrigues FAR, Jimenez PC, Angelim AL, Melo VMM, Rodrigues Filho E, de Oliveira MCF, Costa-Lotufo LV (2012) Cytotoxic activity of fungal strains isolated from the ascidian Eudistoma vannamei. Chem Biodivers 9:2203–2209CrossRefPubMedGoogle Scholar
  46. Morrison-Gardiner S (2002) Dominant fungi from Australia coral reefs. Fungal Divers 9:105–121Google Scholar
  47. Mouchka ME, Hewson I, Harvell CD (2010) Coral-associated bacterial asemblages: current knowledge and the potential for climate-driven impacts. Integr Comp Biol 50:662–674CrossRefPubMedGoogle Scholar
  48. Park MS, Fong JJ, Oh S-Y, Kwon KK, Sohn JH, Lim YW (2014) Marine-derived Penicillium in Korea: diversity, enzyme activity, and antifungal properties. Antonie Van Leeuwenhoek 106:331–345CrossRefPubMedGoogle Scholar
  49. Paul VJ, Ritson-Williams R, Sharp K (2011) Marine chemical ecology in benthic environments. Nat Prod Rev 28:345–387CrossRefGoogle Scholar
  50. Paz Z, Komon-Zelazowska M, Druzhinina IS, Aveskamp MM, Shnaiderman A, Aluma Y, Carmeli S, Ilan M, Yarden O (2010) Diversity and potential antifungal properties of fungi associated with a Mediterranean sponge. Fungal Divers 42:17–26CrossRefGoogle Scholar
  51. Pérez-Portela R, Turon X (2007) Prey preferences of the polyclad flatworm Prostheceraeus roseus among Mediterranean species of the ascidian genus Pycnoclavella. Hydrobiologia 592:535–539CrossRefGoogle Scholar
  52. Pérez-Portela R, Duran S, Palacin C, Turon X (2007) The genus Pycnoclavella (Ascidiacea) in the Atlanto- Mediterranean region: a combined molecular and morphological approach. Invertebr Syst 21:187–205CrossRefGoogle Scholar
  53. Pisut DP, Pawlik JR (2002) Anti-predatory chemical defenses of ascidians: secondary metabolites or inorganic acids? J Exp Mar Biol Ecol 270:203–214CrossRefGoogle Scholar
  54. Rowan R (1998) Diversity and ecology of zooxanthellae on coral reefs. J Phycol 34:407–417CrossRefGoogle Scholar
  55. Schmitt S, Tsai P, Bell J, Fromont J, Ilan M, Lindquist N, Perez T, Rodrigo A, Schupp PJ, Vacelet J, Webster NS, Hentschel U, Taylor MW (2012) Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J 6:564–576PubMedCentralCrossRefPubMedGoogle Scholar
  56. Shenkar N, Swalla BJ (2011) Global diversity of Ascidiacea. PLoS ONE 6:e20657PubMedCentralCrossRefPubMedGoogle Scholar
  57. Sun H-H, Mao W-J, Jiao J-Y, Xu J-C, Li H-Y, Chen Y, Qi X-H, Chen Y-L, Xu J, Zhao C-Q, Hou Y-J, Yang Y-P (2011) Structural characterization of extracellular polysaccharides produced by the marine fungus Epicoccum nigrum JJY-40 and their antioxidant activities. Mar Biotechnol 13:1048–1055CrossRefPubMedGoogle Scholar
  58. Suryanarayanan TS (2012) The diversity and importance of fungi associated with marine sponges. Bot Mar 55:553–564CrossRefGoogle Scholar
  59. Tait E, Carman M, Sievert SM (2007) Phylogenetic divrsity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA). J Exp Mar Biol Ecol 342:138–146CrossRefGoogle Scholar
  60. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729PubMedCentralCrossRefPubMedGoogle Scholar
  61. Tarjuelo I, López-Legentil S, Codina M, Turon X (2002) Defence mechanisms of adults and larvae of colonial ascidians: patterns of palatability and toxicity. Mar Ecol Prog Ser 235:103–115CrossRefGoogle Scholar
  62. Taylor MW, Schupp P, Dahllof I, Kjelleberg S, Steinberg PD (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ Microbiol 6:121–130CrossRefPubMedGoogle Scholar
  63. Thacker RW, Freeman CJ (2012) Sponge-microbe symbioses: recent advances and new directions. Adv Mar Biol 62:57–111CrossRefPubMedGoogle Scholar
  64. Tianero MDB, Kwan JC, Wyche TP, Presson AP, Koch M, Barrows LR, Bugni TS, Schmidt EW (2015) Species specificity of symbiosis and secondary metabolism in ascidians. ISME J 9:615–628CrossRefPubMedGoogle Scholar
  65. Van Bressem M-F, Raga JA, Di Guardo G, Jepson PD, Duignan PJ, Siebert U, Barrett T, de Oliveira Santos MC, Moreno IB, Siciliano S, Aguilar A, Van Waerebeek K (2009) Emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors. Dis Aquat Org 86:143–157CrossRefPubMedGoogle Scholar
  66. Webster NS (2007) Sponge disease: a global threat? Environ Microbiol 9:1363–1375CrossRefPubMedGoogle Scholar
  67. Webster NS, Taylor MW (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol 14:335–346CrossRefPubMedGoogle Scholar
  68. Wheat LJ, Bartlett M, Ciccarelli M, Smith JW (1984) Opportunistic Scopulariopsis pneumonia in an immunocompromised host. South Med J 77:1608–1609CrossRefPubMedGoogle Scholar
  69. White TJ, Bruns T, Lee S, Taylor J (1990) Aplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications. Academic, New YorkGoogle Scholar
  70. Wiese J, Ohlendorf B, Blümel M, Schmaljohann R, Imhoff J (2011) Phylogenetic identification of fungi isolated from the marine sponge Tethya aurantium and identification of their secondary metabolites. Marine Drugs 9:561–585PubMedCentralCrossRefPubMedGoogle Scholar
  71. Williamson B, Tudzynski B, Tudzynski P, Van Kan JA (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8:561–580CrossRefPubMedGoogle Scholar
  72. Xin ZH, Tian L, Zhu TJ, Wang WL, Du L, Fang YC, Gu QQ, Zhu WM (2007) Isocoumarin derivatives from the sea squirt-derived fungus Penicillium stoloniferum QY2-10 and the halotolerant fungus Penicillium notatum B-52. Arch Pharm Res 30:816–819CrossRefPubMedGoogle Scholar
  73. Yarden O (2014) Fungal association with sessile marine invertebrates. Front Microbiol 5:228PubMedCentralCrossRefPubMedGoogle Scholar
  74. Yarden O, Ainsworth TD, Roff G, Leggat W, Fine M, Hoegh-Guldberg O (2007) Increased prevalence of ubiquitous ascomycetes in an acropoid coral (Acropora formosa) exhibiting symptoms of brown band syndrome and skeletal eroding band. Appl Environ Microbiol 73:2755–2757PubMedCentralCrossRefPubMedGoogle Scholar
  75. Yu ZG, Lang G, Kajahn I, Schmaljohann R, Imhoff JF (2008) Scopularides A and B, cyclodepsipeptides from a marine sponge-derived fungus, Scopulariopsis brevicaulis. J Nat Prod 71:1052–1054CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Susanna López-Legentil
    • 1
    Email author
  • Patrick M. Erwin
    • 1
  • Marta Turon
    • 2
  • Oded Yarden
    • 3
  1. 1.Department of Biology and Marine Biology, and Center for Marine ScienceUniversity of North Carolina WilmingtonWilmingtonUSA
  2. 2.Center for Advanced Studies of Blanes (CEAB-CSIC)BlanesSpain
  3. 3.Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and EnvironmentThe Hebrew University of JerusalemRehovotIsrael

Personalised recommendations