Phylogenetic Diversity of Sponge-Associated Fungi from the Caribbean and the Pacific Of

Phylogenetic diversity of sponge-associated fungi from the Caribbean and the Pacific of Panama and theirin vitro effect on angiotensin and endothelin receptors

MARINE BIOTECHNOLOGY

aJessica Bolaños, aLuis Fernando De León, bEdgardo Ochoa, cJoséDarias, dHuzefa A. Raja, eCarolA.Shearer, fAndrew N. Miller, gPatrickVanderheyden,hAndrea Porras-Alfaro, aCatherina Caballero-George

a Institute for Scientific Research and High Technology Services, Bld. 219, City of Knowledge, Clayton, Panama, Republic of Panama

bConservation International, 2011 Crystal Dr. Suite 500, 22202, Arlington, VA, United States

cInstituto de Productos Naturales y Agrobiología (IPNA), Consejo Superior de Investigaciones Científicas, Avda. AstrofísicoFco. Sánchez, 3, 38206 La Laguna, Tenerife, España.

dDepartment of Chemistry and Biochemistry, The University of North Carolina at Greensboro, 435 Sullivan Science Building, 301 McIver Street, Greensboro NC 27412, United States

eDepartment of Plant Biology, University of Illinois, 505 South Goodwin Avenue Urbana, IL 61801, United States

fIllinois Natural History Survey, University of Illinois, 1816 South Oak Street, Champaign, IL 61820, United States

gFaculty of Sciences and Bioengineering Sciences, VrijeUniversiteitBrussel, Building E, Room E7.09, Pleinlaan 2, B-1050 Brussels, Belgium

h Department of Biological Sciences, Western Illinois University, Waggoner Hall 372, 1 University Circle Macomb, IL 61455, United States

*For correspondence –;

Online Resource 2 - Detailed description of fungi associated with specific sponge orders:

Fungi associated with sponges from the order Chondrosida.

Threefungal isolates obtained from sponges from the order Chondrosida collected at two different sites in the Pacific were similar (ID >99%) to MicrosphaeropsisarundinisHQ607976 (Gulf of Panama), Diatrypellasp.FJ800508 (Coiba Island) and Cladosporiumsp.KC339216 (Coiba Island).

Fungi associated with sponges from the order Dictyoceratida.

The sponge Hyatellacfintestinallis was collected in the Pacific around Coiba Island, while the sponge Irciniastrobilina was collected in the Caribbean. Six isolates from H. cfintestinallis were closely related (ID >98%) to AcremoniumimplicatumFN706541, BionectriarossmaniaeAF358227, Cladosporium sp.HM535372, Cochliobolusnisikadoi JN943428, Fusariumsolani HQ379686 and XylariahypoxylonEF423546; and two had low similarity (ID = 93%) to Pleosporales sp. JX264157. Within I. strobilina one isolate was similar (ID 99%) to EutypellascopariaEU702436 and one isolate was distantly related (ID 89%) to its closest match Cosmosporacymosa HQ897828.

Fungi associated with sponges from the order Hadromerida.

Six different genera of sponges from this group were selected. Clionadelitrix, Placospongiaintermedia, Spirastrellasp. and Tethyasp. were collected in the Caribbean; while Laxosuberitessp. and one unidentified specie were collected in the Pacific.The fungal isolate from C.delitrix was similar (ID 100%) to ClonostachysdivergensGU934587. Nine fungal isolates from P.intermedia were closely related (ID >97%) to AcremoniumfurcatumJQ647429,two toAcremoniumimplicatumFN706541, GU189520, one toAcremoniummacroclavatumHQ897806, CosmosporaviliorJN541223, FusariumculmorumKC311482, Hypocreasp.HE584898, HypoxylonmonticulosumJN979435and MicrosphaeropsisarundinisEF094551; an additional isolate was distantly (ID 95%) related to Pleosporaceaesp. HM992501. From the sponge genus Spirastrellasp., twenty six isolates were similar (ID >99%) to their closest match. Four were to AcremoniumimplicatumFN706541, three to BionectriaochroleucaJQ411383, DQ674381, HQ443202, one to Cladosporiumsp.JQ988832, one to Cochliobolussp.JN207295, five were to EutypellascopariaJN601153, HM751800, JQ936179, one to FusariumincarnatumJX045848, one to Hypocreajecorina JN704346, one to Hypocrealixii JQ278697, one to Leptosphaeriaceae sp.FJ884103, one toMicrodiplodiamiyakei HQ248187, two to NectriaipomoeaeJQ717346, one to Penicilliumpaxilli JN617709, one to Massarinasp.JQ889692, one to Preussiasp.HQ607802, one toAscomycota sp. HQ157161,one was 99% similar to Pleosporales sp. JN545763 with a query coverage of 60% and one was distantly related (ID 96%) to Pleosporales sp. HQ649968. In Tethyasp. one isolate was closely related to Eutypellascoparia JQ936179. In sponges from the Suberitidae family, one isolate was closely related (ID >99%) to AcremoniumimplicatumGU189520, two to Arthriniumsp.AY513945, two isolates were to CochlioboluslunatusHQ607991, one to Biscogniauxiasp.FJ884075, and one to Fusariumsp.JN092584.

Fungi associated with sponges from the order Halichondrida.

Two genera collected in the Pacific represented this order. Within the genus Axinella thirty four isolates were closely related (ID >97%) to their top match. Eight isolates were similar to AcremoniumimplicatumFN706541,two to AcremoniumstrictumGU219467, one to Arthriniumphaeospermum KC253945, one to Arthriniumsp.JF817255, two to Ascomycotasp. EF060480, one to BionectriaochroleucaDQ279793, one to Cladosporiumsp.HM535372, one to Cochlioboluslunatus HQ607991, one to Coprinellussp.JN418793, one to CordycipitaceaeKC311493, one to EutypellascopariaJQ936179, one to Fusariumsolani JQ723751, two to Fusariumsp. JN092584,GU233852, one to Hypocreajecorina JN704346, one to Hypocrealixii FR872742, one to LeptosphaerulinachartarumJN862799, one to Microsphaeropsisarundinis FJ798603, one to Nectria sp. HQ130677,one to Nigrosporaoryzae JF909550, two to Sordariomycetessp. FJ799945, one to Sordariomycetessp. FJ799946 andtwo to Xylariasp. JX624317, HM535394. Five isolate showed low similarity (ID 92-96%) to Arthriniumphaeospermum HQ914942, Cochliobolussp. HQ608077, Harknessiarhabdosphaera DQ923532, Pleosporales sp. JX264157, Xylariaceaesp. EU009990, and two more isolates were distantly related to Sordariomycetessp. JQ759196 (ID 83%) and to Xylariasp. JQ862705 (ID 89%).

Within the sponge genus Scopalinasp. two isolates were similar (ID 99%) to AcremoniumimplicatumFN706541 and to Preussiasp. JN159709, while the third one was distantly related (ID 92%) to Acremoniumsp.AY303601.

Fungi associated with sponges from the order Haplosclerida.

Within the genus Amphimedoncompressa, collected in the Caribbean, two isolates were similar (ID >99%) LetendraeahelminthicolaJQ026217, PleosporalesKC248544; two more isolates showed low similarity (ID 95-96%) Beauveriasp. HQ880791, Cosmosporavilior JN541223. Also, from the Caribbean, the sponge genus Amphimedonviridis was obtained and two fungal isolates were found similar (ID 99%) to Eutypellascoparia JX049395 and Sarocladiumstrictum JQ717318. Seventeen fungal isolates obtained from the sponge genus Haliclona were similar (ID 97-100%) to Ascomycotasp.HQ607850,Acremoniumstrictum GU219467, Bionectriapityrodes AY254158,two to Cladosporiumsp.KC145175, EF029809,Hypocreasp.HQ630960, two to Hypocreajecorina JN704346, Hypocrealessp.JQ905759, Nectriaipomoeae JQ717346, three to Pleosporales sp.EF694666, two to Pleosporales sp.HQ832823, one toPleosporales sp. JX264157 and one to Pleosporales sp. JN851029. Two more isolates were distantly related (ID 95-96%) to Ascomycota sp.HQ731636, one to Eutypellascoparia EU702437, one to Hypocreales sp.HQ607982, one to Phellinusbicuspidatus AY558610 and one toXylariaceae sp. AY315402. Additionally, four isolates were distantly related (ID <90%) to Ascomycota sp. FJ556910, Acremoniumsp.HQ631064, Coprinopsissp. AB564721, and Sordariomycetes sp.JQ760354. Forty fungal isolates obtained from the sponge genus Niphateserecta were similar (ID >97%) to Acremoniumimplicatum FN706541 (four isolates), Albonectriarigidiuscula HM054153,Annulohypoxylonstygium EU272517, Ascomycota sp. EF060480 (two isolates), Beauveriasp.JQ266171, Bionectriaochroleuca DQ279793, Cosmosporavilior JN541223, Cytosporasp.DQ993620, DQ092502, Cytosporarhizophorae DQ996040, Diaporthesp.FJ037739, Diatrypaceae sp.JF773650, Diatrypellapulvinata JX427039, Dothideomycetes sp. JQ905824, Eutypellascoparia JQ936179 (three isolates), EU702437 (one isolate), JX049395 (one isolate), Fusarium sp.HQ696081, Fusariumsolani GU355662, FusicladiumafricanumEU035424, Hypocreales sp.EF060508, Microsphaeropsisarundinis JQ647902, Nectriaipomoeae AB513849, Pleosporales sp. JN559399 (two isolates), Pleosporales sp. HQ631015, Pleosporales sp. HM116750, Pleosporales sp. JX264157, Pleosporales sp.GQ254685, Pseudallescheria sp. AB734453, Sordariomycetes sp. JQ761017, Xylariaceae sp. EU010004, Xylaria sp. EU009989 and one more isolate was similar to Basidiomycota sp.HQ022513. Six isolates within the same sponge were distantly related (ID 90-96%) to Ascomycota sp. GU985214, Bionectriasp.GU166499, Diatrypellasp. FJ800508, Eutypellascoparia EU702434, Hypocrealessp.FJ375172 and Basidiomycota sp. HQ022514. One additional isolate obtained from an unidentified genus of sponge from the family Niphatidae was distantly related to Pleosporales sp.EU049289 (ID 91%).

Within the Xestospongia genus, ten isolates were similar (ID >98%) to Cytosporasp.DQ993620, Cytosporarhizophorae DQ996040, Eutypellascoparia JN601153 (two isolates), JQ936179 (three isolates), FusariumsolaniHM992494, JX241656 and Pleosporales sp.EF694666. Four isolates were distantly (ID 92-96%) related to Acremoniumimplicatum HQ914930, Eutypellascoparia EU702437 (two isolates) and Pleosporales sp. HQ832823.

Fungi associated with sponges from the order Homosclerophorida.

The sponge Plakinastrellaonkodes, collected in two different sites in the Caribbean coast of Panama, yield very few fungal isolates. One isolate was closely related to EutypellascopariaEU702436 (ID 99%) and two were distantly related to Herpotrichiellaceae sp.HE605219 (ID 93%) and Hypocreales sp.GU017486 (ID 96%). Within the sponge Aka coralliphagum one isolate was found similar to Hypocreales sp. GU595042 (ID 99%).

Fungi associated with sponges from the order Poecilosclerida.

Six different species of sponges from this order were collected for this study. Within the sponge specie Lissondendoryxcolombiensis, collected in the Caribbean, ten isolates were similar (ID > 97%) to Acremoniumimplicatum FN706541, Ascomycota sp.EF060480, Colletotrichumsp.JX009429, Hypocrealixii JF923807, Hypocreasp. JQ988834, Eurotiales sp. HQ207033, Myrotheciumsp.AJ301998, Pleosporales sp.JN559399,Pleosporales sp. EF694666 and Xylariaceae sp.EU010001. Three more isolates were distantly related to Ascomycete sp.EF632079 (ID 92%), Ascomycota sp. FJ375137 (ID 92%) and Hypocreales sp.EF060755 (ID 96%). One isolate obtained from the sponge Iotrochotabirotulata had low similarity (ID 78%) to the 68% of the sequence of its closest match, EutypellakochianaAJ302462.

Within the Caribbean sponge Desmapsanaanchorata five isolates were similar (ID 98-99%) to AcremoniumstrictumGU219467, Albonectriarigidiuscula KC771494,Ascomycete sp. EU002909, Pleosporales sp. KC339242 and Xylariaceae sp. AB440109. Two more isolates were distantly related to Pleosporales sp. HM116750 (ID 89%) and Hypocreales sp. KC007132 (ID 85%). Moreover, within the sponge genus Mycale sp., seven isolates were similar (ID > 97%) to Ascomycota sp. EF060480, Cosmospora sp. JN995632, Hypocreasp. JQ775562, Pleosporales sp. EF694666, Pleosporales sp. HQ832823, Xylariasp. JQ862705 and Arthopyreniaceae sp. DQ682563. Four isolates within this sponge were distantly related to Hypocreasp.JQ988821 (ID 96%), Xylariaceae sp.JF773597 (ID 96%), Hypocreales sp.HQ248209 (ID 88%) and Hypocreales sp.HQ248209 (ID 87%).

In the Raspaillidae family, five unidentified specimens of sponges were collected in the Pacific, within this sample nine were similar (ID >98%) to Cladosporium sp.JQ988832, Hypocreales sp.GU017508, Hypocreales sp.EF060508, Hypoxylon sp.KC178664 (two isolates), Pleosporales sp.JX264157, Xylariaceae sp.AB741622, Xylariaceae sp.AB741621 and Xylariaceae sp.KC178661. Two isolates showed low similarity to Pleosporales sp.JF288550 (ID 93%) and Xylariaceae sp. AB440133 (ID 90%).

Within the sponge specie Tedaniaignis, AnnulohypoxylonstygiumKC771510, Aspergillus sp.JQ717355, Cytospora sp.DQ993620, Diaporthe sp.FJ037739, Dothideomycetes sp.JQ905824, Fusarium sp. FJ158122, AF178402, Hypocreales sp. HQ649882, EF060553 (two isolates), Pleosporales sp.JX264157, Pleosporales sp. HQ832823, Xylariales KC181931 and Xylariaceae sp. HQ117857. Seven isolates were distantly related to Agaricales sp. GQ923956 (ID 89%), HypocrealesKC311472 (ID 91%, two isolates), HQ248209 (ID 87%), Diatrypaceae sp. JF773650 (ID 90%), Pleosporales sp. HQ631051 (ID 94%) and Xylariaceae sp.EU010001 (ID 96%).

Fungi associated with sponges from the order Spirophorida.

Twelve fungal isolates were obtained from the sponge Cynachirellaalloclada collected in the Caribbean. Nine of which were similar (ID >97%) to Coprinellussp.JN418793, Fusariumsp.AF178422, Hypocreales sp.EF060508, Hypocreales sp.EF060508, Leptosphaerulinachartarum JN862799, Letendraeahelminthicola JQ026217, Phomasp.JQ621876, Pseudallescheriasp. DQ682599 and Xylariaceae sp.EU010001. Three more isolates were distantly related to Acremonium sp.AY303601 (ID 92%), DidymellaexitalisKC311477 (ID 90%) and Pleosporales sp. HQ832823 (ID 94%).

Fungi associated with sponges from the order Verongida.

Two main genera of sponges namely Aplysina and Verongula, were collected in both the Caribbean and the Pacific of Panama. Within the sponge Aplysinacauliformis thirty four isolates were closely related (ID >98%) to Acremoniumimplicatum FN706541,Biscogniauxia sp.FJ884075, Coprinellus radians HM595561, CoprinellusdisseminatusKC771477, Eutypellasp.JN637945, Eutypellascoparia EU702436, Diatrypaceae sp. JF773650, Diatrypellapulvinata JX427039, Hypocrealixii JF923807, Hypocreales sp. JQ905656, Hypoxylonsp. DQ322114, Lasiodiplodiasp. JN615242, Microsphaeropsisarundinis AB775571, Nectriaipomoeae JQ717346 (two isolates), Pestalotiopsis sp. JX684005, KC203051, Phomopsissp. JQ905726, Pleosporales KC248544, HQ649851, HQ108019, EF694666,HQ832823, Trichodermasp. DQ164408, HQ630998, AY154920,Xylariaceae sp. AB741621, AB741619 and Xylariasp.EU009958 (two isolates), EU009989 (three isolates), HQ107995 and Xylariaceae sp. EU009990. Six more isolates were distantly related (ID 91-95%) to Biscogniauxiacapnodes EF026131, Dothideomycetes sp.GQ153059, HypocrealesKC311472, Pleosporales sp. EF694666,Pleosporales sp. AB255299 and Xylariaceae sp. EU010003.Two isolates showed low similarity to Hypocreales sp.JQ905658 (ID 86%) and Pleosporales sp.JX264157 (ID 90%). Within the sponge Aplysinachiriquensis, collected in the Pacific, four isolates were similar (ID >99%) to Arthriniumsp.HQ832842, Cladosporiumsp.JQ988832 (two isolates) and Eutypellasp.FJ800515. In the Caribbean, the sponge Aplysinafulva was collected and twenty nine isolates showed close similarity (ID >97%) to Acremoniumimplicatum KC464332, FN706541, Acremoniumstrictum GU219467, Beauveriasp.JQ266171, Diatrypellapulvinata JX427039, Dothideomycetes sp. JQ905824 (two isolates), Eutypellascoparia JQ936179, Eutypellascoparia EU702436 (four isolates), Hypocreasp. HQ657316, JQ988834, HQ657295, Hypocreasp.JQ988820 (two isolates), Hypocrealixii JX082390 (two isolates), Hypocreales sp.JQ905673, Massarinasp.JQ889692 (two isolates), Pestalotiopsissp.KC540782, Pleosporales sp. HQ832823, JN559399, Pleosporales sp.JX264157, Plectosporium sp.AB685487, Sordariomycetes sp. JQ760257, Xylariales KC181931, Xylareaceae EU010001 and Fungal sp. JQ747679. Four additional isolates were distantly related to Eutypellacerviculata AJ302461, Hypocreales sp.HQ248209, NigrogranamackinnoniiKC288117, Wrightoporiatropicalis FJ904857. Within the same specie two more isolates showed low similarity to Xylariales sp.AB511813 (ID 84%) and Hypocreales sp.DQ350128 (ID 89%). Within Aplysinagerardogreeni twelve isolates were closely related (ID >97%) to Ascomycete sp.AJ279474, Arthriniumsp.JN618364, Eutypellasp.FJ800515, Eutypellascoparia JQ936179, JN601153, EU702436, JX049395, Fusariumsolani JN411816, Pleosporaceae EF687932, Pleosporales sp. JN559399, Sordariomycetes JQ760019 and Xylariaceae sp. AB440109. Five additional isolates were distantly related (ID 91-96%) to Ascomycete EF632079,EutypellascopariaEU702437, Hypocreales KC311472, Hypocreales sp. GU017508 and Xylariaceae JX298902. Four more isolates showed low similarity to Ascomycete sp.EF632079 (ID 90%), Hypocreales sp.GU017486 (ID 83%) and WhalleyamicroplacaFJ416301 (ID 81%), JX914486 (ID 80%).

Within three unidentified sponge species from the genus Aplysina collected in the Caribbean twenty seven isolate were similar (ID > 98%) to Annulohypoxylonstygium KC771510, Beauveriabassiana KC551951, Diaporthesp.EF423521 (two isolates), FJ799939, Dothideomycetes sp.JQ905824 (two isolates), Eutypellascoparia JX049395, EU702436, JQ936179 (two isolates), JN601153 (two isolates), Eutypellasp. JN637945 (two isolates), Pleosporales sp. HQ832823 (three isolates), JF288550, EF694666 (two isolates), Phomasp. JQ621876 (two isolates), Rhytidhysteronrufulum HQ832843, and Xylariaceae sp.JN411813, EU010001, AB440109. Ten additional isolates were distantly related (ID 91-95%) to HypocrealesKC311472 (two isolates), HQ607953, Marasmiusexustoides JN601434,Pleosporales sp. KC771509, JF288550 (three isolates), Xylariaceae sp. AB440101 and Xylariasp. DQ780448.

Within twelve unidentified sponge species from the genus Aplysina collected in the Pacific twenty seven isolates were closely related (ID >97%) to Annulohypoxylonsp.KC147571, Acremoniumsp.JQ717349, Corynesporacassiicola KC662096, Diatrypaceae sp.JF773650, Dothideomycete sp. EU680515, EU680530, Eutypellascoparia EU702437, Eutypellasp.FJ800515, Eutypellasp.JN637945, Guignardiasp. HQ622105, Hypocreales sp. EF060726, Myrotheciumsp.JQ936269, Pestalotiopsis sp.JX684003, Phomasp.JQ621876, Pleosporales sp. GQ254685 (two isolates), EF694666, Rhytidhysteronrufulum EU020026, Sordariomycetes JQ760992, JQ760019, EF694652, Strelitzianaafricana DQ885895, Trichodermasp. JQ655465, Westerdykellanigra AY943049, Fungal endophyte sp. EU686045 and Fungal sp. EU558536, JN098081, JQ747679. Two isolates show low similarity to Ascomycota sp.GU566290 (ID 91%) and Pleosporales sp. GU595040 (ID 82%).

In the sponge Verongulareiswigi collected in the Caribbean, twenty five isolates were similar (ID >97%) to Diaporthesp. FJ799937 (two isolates), GU066726 (two isolates), Eutypellascoparia JN601153, JQ936179 (two isolates), Eutypellasp.JN637945, Hypocreales sp.JQ905649, Nectriamariannaeae GU586835, Pleosporales sp.EF694666 (six isolates), JN559399, HQ832823, Pseudallescheria sp.GU827497, Phellinustropicalis AF534077, Pseudallescheriaboydii KC800585, Phomasp. JQ621876, JX868743, Fungal sp.JQ747679 and Fungal sp.FJ439590. Six isolates were distantly related (ID 91-95%) to Ascomycota sp.GU985214, Diatrypellapulvinata FR715523, Hypocreales EF060755, KC311472 (two isolates) and Xylariaceae sp. EU010003.

Within eightunidentified sponge species collected in the Caribbean twenty seven isolates were closely related (ID >98%) to Acremoniumsp.JQ717349, Ascomycota sp. HM052813, Biscogniauxiasp. FJ884075, Cladosporiumsp.JQ988832, Eutypellascoparia JQ936179 (five isolates), EU436688, Eutypella sp.EF488380, Fusariumsp. JX891784, Hypocreales sp. EU164804, Hypocreasp. HE608774, Letendraeahelminthicola JQ026217, NectriaipomoeaeJQ717346, Pestalotiopsissp. KC837100 (two isolates), JF304634, Phomasp.JQ621876, Sordariomycetes sp. JX298932, Xylariacubensis GU373810, Xylariaceae sp. AB440109, AB741620, Fungal sp. EU558536 and Fungal endophyte EU977299. Nine isolates were distantly relatated (ID 91-96%) to Ascomycota sp. GU985214, Diatrypellapulvinata FR715523, Eutypapetrakiivar. petrakii AJ302456, Eutypellascoparia EU702437, Eutypellasp.JQ717311, Hypocreales sp.EF060755, DQ682584 and Pleosporales KC248544, HQ649992. Three isolates showed low similarity to Ascomycete sp. EF632079 (ID 89%), Hypocreales sp.JN851014 (ID 88%) and Xylariales sp.HQ823756 (ID 83%).

Within six unidentified sponge species collected in the Pacific twelve isolates were similar (ID >98%) to Acremoniumsp. JQ717349 (two isolates), Annulohypoxylonstygium KC771510, Ascomycota sp. HQ636419, Hypocreasp.HQ657316, JQ988820, Massarinasp. JQ889692, Plectosporiumsp. AB685487, Pleosporales sp. EF694666, Xylariasp. DQ631944, Basidiomycota sp.HQ022513, Fungal sp.JQ747679. Six isolates were distantly related (ID 91-96%) to Diatrypellapulvinata FR715523, Hypocreales sp EF694655, Hypocreasp.KC311472, Pleosporales KC248544, KC771509, JN545763. Seven more isolates showed low similarity to Ascomycota sp. GU017493 (ID 90%), Cystostereummurrayi JQ358801 (ID 87%), Coniothyriumfuckelii HQ832837 (ID 89%), Munkovalsariaappendiculata DQ435529 (ID 90%), Pleosporales sp. HM992807 (ID 87%), JN859348 (ID 78%) and Xylariaceae sp. AB741596 (ID 83%).

Overall, thirty eight isolates showed very low similarity (ID 90%) to their closest match, the majority of which were found in the genus Aplysinasp(Appendix 1).

1