Phylogenetic Assignment of the Fungicolous (Ascomycota, Xylariales) and Investigation of its Secondary Metabolites.

mic oo ganisms A icle Phylogene ic Assignmen o he Fungicolous Hypoxylon in adens (Ascomyco a, Xyla iales) and In es iga ion o i s Seconda y Me aboli es Ke in Becke 1,2 , Ch is ophe Lambe 1,2,3 , Jö g Wieschhaus 1and Ma c S adle 1,2,* 1Depa men o Mic obial D ugs, Helmhol z Cen e o In ec ion Resea ch GmbH (HZI), Inho ens aße 7, 38124 B aunschweig, Ge many; [email p o ec ed] (K.B.); ch is ophe [email p o ec ed] (C.L.); [email p o ec ed] (J.W.) 2Ge man Cen e o In ec ion Resea ch Associa ion (DZIF), Pa ne si e Hanno e -B aunschweig, Inho ens aße 7, 38124 B aunschweig, Ge many 3Depa men o Molecula Cell Biology, Helmhol z Cen e o In ec ion Resea ch GmbH (HZI) Inho ens aße 7, 38124 B aunschweig, Ge many *Co espondence: [email p o ec ed]; Tel.: +49-531-6181-4240; Fax: +49-531-6181-9499 Recei ed: 23 July 2020; Accep ed: 8 Sep embe 2020; Published: 11 Sep embe 2020   Abs ac : The ascomyce e Hypoxylon in adens was desc ibed in 2014 as a ungicolous species g owing on a membe o i s own genus, H. agi o me, which is conside ed a a e li es yle in he Hypoxylaceae. This ende sH. in adens an in e es ing a ge inou e o s o ind new bioac i e seconda yme aboli es om membe s o he Xyla iales. So a , only ola ile o ganic compounds ha e been epo ed om H. in adens, bu no in es iga ion o non- ola ile compounds had been conduc ed. Fu he mo e, a phylogene ic assignmen ollowing ecen ends in ungal axonomy ia a mul iple sequence alignmen seemed p ac ical. A cul u e o H. in adens was hus subjec ed o subme ged cul i a ion o in es iga e he p oduced seconda y me aboli es, ollowed by isola ion ia p epa a i e ch oma og aphy and subsequen s uc u e elucida ion by means o nuclea magne ic esonance (NMR) spec oscopy and high- esolu ion mass spec ome y (HR-MS). This app oach led o he iden i ica ion o he known la iolin ( 1 ) and 3,3-bi la iolin ( 2 ) as he main componen s, which had ne e been epo ed om he o de Xyla iales be o e. Assessmen o hei an imic obial and cy o oxic e ec s ia a panel o commonly used mic oo ganisms and cell lines in ou labo a o y did no yield any e ec s o ele ance. Concu en ly, genomic DNA om he ungus was used o cons uc a mul igene phylogeny using ibosomal sequence in o ma ion om he in e nal ansc ibed space egion (ITS), he 28S la ge subuni o ibosomal DNA (LSU), and p o einogenic nucleo ide sequences om he second la ges subuni o he DNA-di ec ed RNA polyme ase II (RPB2) and β - ubulin (TUB2) genes. A placemen in a newly o med clade wi h H. ugodes was s ongly suppo ed in a maximum-likelihood (ML) phylogeny using sequences de i ed om well cha ac e ized s ains, bu he exac posi ion o said clade emains unclea . Bo h, he chemical and he phylogene ic esul s sugges u he inqui ies in o he li es yle o his unique ungus o ge a be e unde s anding o bo h, i s ecological ole and unc ion o i s p oduced seconda y me aboli es hi he o unique o he Xyla iales. Keywo ds: Ascomyco a; ch oma og aphy; mul igenephylogeny; naph hoquinones; axonomy; Xyla iales 1. In oduc ion Hypoxylon is he la ges genus wi hin he ungal amily Hypoxylaceae (o de Xyla iales, Ascomyco a) wi h mo e han 230 desc ibed species [ 1 ]. The s oma a ( ui ing bodies) o Hypoxylon a e o en a he conspicuous and can equen ly be ound on dead wood in all o es ed a eas o he wo ld, bu he mycelia o hese ungi a e also among he mos equen ly isola ed endophy es o seed plan s. This unde lines he ecological impo ance o he genus [ 2 , 3 ]. Recen axonomical Mic oo ganisms 2020,8, 1397; doi:10.3390/mic oo ganisms8091397 www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2020,8, 1397 2 o 14 ad ances e ol ed on he eo ganiza ion o Hypoxylon and allies, using a polyphasic concep combining mo phological, molecula phylogene ics wi h special emphasis on mul i-locus genealogies and chemo axonomic app oaches. This ul ima ely led o he esu ec ion o he Hypoxylaceae i sel and he seg ega ion o gene a like Hypomon agnella and Jack oge sella [ 2 , 3 ], while inc easing chemo axonomical knowledge suppo ed he ea men o gene a like Annulohypoxylon as an own genus whe e sequence in o ma ion de i ed om he widely used In e nal ansc ibed space egion (ITS) alone did no gi e a conclusi e esolu ion [ 4 , 5 ]. Membe s o he Hypoxylacaeae a e also known o p oduce an as onishing amoun o di e se seconda y me aboli es, making hem aluable a ge s o na u al p oduc esea che s [ 6 ]. This esea ch b anch is u he suppo ed by he inc easing numbe o high-quali y genomes a ailable [ 7 ], expanding he classical sc eening app oaches o na u al p oduc chemis y by he possibili y o genome mining and biosyn hesis s udies o p omising bioac i e compounds. The mono ypic species Hypoxylon in adens was ecen ly epo ed om F ance as a ungicolous species g owing on s oma a o a membe o i s own genus, H. agi o me [ 8 ]. Only a ew cases o his appa en ly mycophilic li es yle a e known om he genus Hypoxylon and o he gene a ha now belong o he Hypoxylaceae (i.e., Chlo os oma and Thuemenella [ 9 ], o which no mycelial cul u es a e p esen ly a ailable in he public domain), and i was no possible o e alua e whe he he s oma a jus g ow coinciden ally on he s oma a o hei ela i es, o whe he he e is some o m o pa asi ism o mu ualism in ol ed. We we e able o ob ain a cul u e om he ascospo es o he holo ype specimen o H. in adens ha can now be s udied in-dep h o i s physiological and ecological ai s, including seconda y me aboli e p oduc ion. In a p e ious s udy we ha e checked he cul u es o H. in adens and H. mac oca pum [ 8 ], and he only epo ed me aboli es om hose species a e se e al ola ile o ganic compounds (VOC) [ 10 , 11 ] ha we e in es iga ed by GC-MS analysis and o al syn hesis a e obse a ion o a s ong odo o igina ing om mycelia g own on oa meal aga [8]. The cu en s udy is dedica ed o assessing he phylogene ic a ini ies o H. in adens ia a ou -locus phylogeny, as well as he in es iga ion o i s seconda y me aboli es, ollowed by e alua ion o he cy o oxici y and an imic obial ac i i y o he newly isola ed compounds. 2. Ma e ials and Me hods 2.1. Gene al Elec osp ay mass (ESI-MS) spec a we e eco ded wi h an Ul iMa e ® 3000 Se ies UHPLC (The mo ishe Scien i ic, Wal man, MA, USA) u ilizing a C18 Acqui y ® UPLC BEH column (2.1 × 50 mm, 1.7 µ m; Wa e s, Mil o d, MA, USA), connec ed o an amaZon speed ® ESI-Ion ap-MS (B uke , Bille ica, MA, USA). HPLC pa ame e s we e se as ollows: sol en A: H 2 O+0.1% o mic acid, sol en B: ace oni ile (ACN)+0.1% o mic acid; g adien : 5% B o 0.5 min, inc easing o 100% B in 19.5 min, keeping 100% B o u he 5 min; low a e 0.6 mL/min, DAD de ec ion 200−600 nm. High esolu ion elec osp ay mass (HR-ESI-MS) spec a we e ob ained wi h an Agilen 1200 In ini y Se iesHPLC (Agilen Technologies, San aCla a, CA,USA) connec ed o amaXis ® elec osp ay ime-o - ligh mass spec ome e (ESI-TOF-MS; B uke ; HPLC condi ions same as o ESI-MS measu emen s). Nuclea magne ic esonance (NMR) spec a we e eco ded wi h an A ance III 500 spec ome e (B uke , 1 H NMR: 500 MHz, 13 C NMR: 125 MHz). UV/ is spec a we e aken wi h a UV-2450 spec opho ome e (Shimadzu, Kyo o, Japan). 2.2. Fungal Ma e ial and Molecula Phylogeny A cul u e om he holo ype specimen o Hypoxylon in adens was used in his s udy. A well-g own cul u e o H. in adens on YM 6.3 aga was used o genomic DNA (gDNA) ex ac ion using a comme cially a ailable ki as desc ibed by Kuhne e al. [ 12 ]. The ob ained gDNA was used o ampli y ou di e en loci: he in e nal ansc ibed space egion (ITS), he 28S la ge subuni o ibosomal DNA (LSU), he second la ges subuni o he DNA-di ec ed RNA polyme ase II (RPB2) and β - ubulin Mic oo ganisms 2020,8, 1397 3 o 14 (TUB2) wi h p ime s as desc ibed elsewhe e [ 13 – 17 ]. PCR p og ams, subsequen amplicon pu i ica ion and sequencing we e conduc ed as desc ibed p e iously [9,18]. Sequence assembly and analysis was done ollowing Wend e al. [ 2 ] wi h he p og am package Geneious ® 7.1.9 (h p://www.geneious.com, [ 19 ]). Taxon selec ion and complemen a ion o sequences was based on he da ase o Si e al. [ 20 ], which comp ised many ype s ains, as well as au hen ic ep esen a i es o all phylogene ic lineages o he genus Hypoxylon and allied Hypoxylaceae. The GenBank accession numbe s o he sequences used a e gi en in Table 1. Loci we e aligned using he in-buil e sion o he p og am MAFFT 7.017 wi h se ings o he G-INS-I algo i hm se o de aul [ 21 ]. The esul ing alignmen was il e ed o phylogene ic in o ma i e posi ions ia he se e e sion o Gblocks (Cas esana Labs, [ 22 ]), low s ingency se ings wi h op ions o smalle inal blocks and gap posi ions wi hin inal blocks, see Tala e a and Cas esana [ 23 ] and Wend e al. [ 2 ]. Molecula phylogene ic in e ence ollowed using he clien e sion o IQT ee (Ve sion 2.0-RC2, [ 24 ]) wi h a pa i ioned da ase [ 25 ] and ModelFinde o model selec ion [ 26 ]. Non-pa ame ic boo s ap (BS, ≥ 50%, majo i y consensus [ 27 ]) and Shimodai a-Hasegawa app oxima e likelihood- a io- es (SH-aLRT, ≥85% [28,29]) alues we e assigned o he bes sco ing maximum-likelihood (ML) ee. Mic oo ganisms 2020,8, 1397 4 o 14 Table 1. Sequence accession numbe s, co esponding isola es, and li e a u e e e ence o sequences used o he molecula phylogene ic in e ence. Species- ype specimen a e gi en he co esponding label (HT =holo ype; ET =Epi ype). The newly sequenced H. in adens isola e is highligh ed in bold. Species S a us S ain O igin GenBank Acc. No. o DNA Loci Re e ences ITS LSU RPB2 TUB2 Annulohypoxylon annula um ET CBS 140775 Texas KY610418 KY610418 KY624263 KX376353 [2,30] A. annula um DSM 107931 Texas MK287534 AJ390397 MK287546 MK287559 MK287572 [20] A. mo i o me CBS 123579 Ma inique KX376321 KY610425 KY624289 KX271261 [2,31] A. ni ens MFLUCC 12.0832 Thailand KJ934991 KJ934992 KJ934994 KJ934993 [32] A. s ygium MUCL 54601 F ench Guinea KY610409 KY610475 KY624292 KX271263 [2] A. unca um ET CBS 140778 Texas KY610419 KY610419 KY624277 KX376352 [2,30] A. unca um DSM 107925 Texas MK287531 MK287543 MK287556 MK287569 [20] Daldinia concen ica CBS 113277 Ge many AY616683 KY610434 KY624243 KC977274 [2,12,33] D. eschschol zii MUCL 45435 Benin JX658484 KY610437 KY624246 KC977266 [2,12,34] D. pe iniae ET MUCL 49214 Aus ia AM749937 KY610439 KY624248 KC977261 [2,12,35] D. placen i o mis MUCL 47603 Mexico AM749921 KY610440 KY624249 KC977278 [2,12,35] En onaema liquescens ATCC 46302 USA KY610389 KY610443 KY624253 KX271248 [2] G aphos oma pla ys oma HT CBS 270.87 F ance JX658535 DQ836906 KY624296 HG934108 [2,34,36,37] Hypom. ba ba ensis HT STMA 14081 A gen ina MK131720 MK131718 MK135891 MK135893 [3] Hypom. mon iculosa ET MUCL 54604 F ench Guiana KY610404 KY610487 KY624305 KX271273 [2] Hypom. submon iculosa CBS 115280 F ance KC968923 KY610457 KY624226 KC977267 [2,12] Hypoxylon ca neum MUCL 54177 F ance KY610400 KY610480 KY624297 KX271270 [2] H. ce cidicola CBS 119009 F ance KC968908 KY610444 KY624254 KU684189 [2,12] H. c oceum DSM 107926 Texas MK287535 MK287547 MK287560 MK287573 [20] H. c oceum DSM 107932 Texas MK287532 MK287544 MK287557 MK287570 [20] H. c ocopeplum CBS 119004 F ance KC968907 KY610445 KY624255 KC977268 [2,12] H. endle i MUCL 54792 F ench Guinea KF234421 KY610481 KY624298 KF300547 [2,12] H. agi o me ET MUCL 51264 Ge many KC477229 KM186295 MK887342 KX271282 [2,20,32,38] H. uscum ET CBS 113049 F ance KY610401 KY610482 KY624299 KX271271 [2] H. g iseob unneum T CBS 331.73 India KY610402 KY610483 KY624300 KC977303 [2,12] H. guilanense HT MUCL 57726 I an MT214997 MT214992 MT212235 MT212239 [39] H. haema os oma ET MUCL 53301 Ma inique KC968911 KY610484 KY624301 KC977291 [2,12] H. howeanum MUCL 47599 Ge many AM749928 KY610448 KY624258 KC977277 [2,12,35] H. hypomil um MUCL 51845 Guadeloupe KY610403 KY610449 KY624302 KX271249 [2] Mic oo ganisms 2020,8, 1397 5 o 14 Table 1. Con . Species S a us S ain O igin GenBank Acc. No. o DNA Loci Re e ences ITS LSU RPB2 TUB2 H. in adens HT MUCL 51475 F ance MT809133 MT809132 MT813037 MT813038 This S udy H. in es iens CBS 118183 Malaysia KC968925 KY610450 KY624259 KC977270 [2,12] H. la e ipigmen um HT MUCL 53304 Ma inique KC968933 KY610486 KY624304 KC977290 [2,12] H. leno mandii CBS 119003 Ecuado KC968943 KY610452 KY624261 KC977273 [2,12] H. lienhwacheense MFLUCC 14-1231 Thailand KU604558 MK287550 MK287563 KU159522 [20,40] H. musceum MUCL 53765 Guadeloupe KC968926 KY610488 KY624306 KC977280 [2,12] H. oli aceopigmen um T DSM 107924 Texas MK287530 MK287542 MK287555 MK287568 [20] H. papilla um HT ATCC 58729 USA KC968919 KY610454 KY624223 KC977258 [2,12] H. pe o a um CBS 115281 F ance KY610391 KY610455 KY624224 KX271250 [2] H. pe iniae HT CBS 114746 F ance KY610405 KY610491 KY624279 KX271274 [2,30] H. pilge ianum STMA 13455 Ma inique KY610412 KY610412 KY624308 KY624315 [2] H. po phy eum CBS 119022 F ance KC968921 KY610456 KY624225 KC977264 [2,41] H. pulicicidum HT CBS 122622 Ma inique JX183075 KY610492 KY624280 JX183072 [2,42] H. ickii ET MUCL 53309 Ma inique KC968932 KY610416 KY624281 KC977288 [2,12] H. ubiginosum ET MUCL 52887 Ge many KC477232 KY610469 KY624266 KY624311 [2,38] H. samuelsii ET MUCL 51843 Guadeloupe KC968916 KY610466 KY624269 KC977286 [2,12] H. exense T DSM 107933 Texas MK287536 MK287548 MK287561 MK287574 [20] H. icinense CBS 115273 F ance JQ009317 KY610471 KY624272 AY951757 [2,4] H. ugodes ET MUCL 54794 S i Lanka KF234422 KY610493 KY624282 KF300548 [2,12] H. ogesiacum CBS 115273 F ance KC968920 KY610417 KY624283 KX271275 [2,12] Jack oge sella cohae ens CBS 119126 Ge many KY610396 KY610497 KY624270 KY624314 [2] J. minu ella CBS 119015 Po ugal KY610381 KY610424 KY624235 KX271240 [2] J. mul i o mis ET CBS 119016 Ge many KC477234 KY610473 KY624290 KX271262 [2,12,30] Py enopolypo us hun e i ET MUCL 52673 I o y Coas KY610421 KY610472 KY624309 KU159530 [2,30] P. laminosus HT MUCL 53305 Ma inique KC968934 KY610485 KY624303 KC977292 [2,12] P.nica aguensis CBS 117739 Bu kina Faso AM749922 KY610489 KY624307 KC977272 [2,12,35] Rhopalos oma angolense CBS 126414 I o y Coas KY610420 KY610459 KY624228 KX271277 [2] Thamnomyces dend oidea HT CBS 123578 F ench Guinea FN428831 KY610467 KY624232 KY624313 [2,43] Xyla ia hypoxylon ET CBS 122620 Sweden KY610407 KY610495 KY624231 KX271279 [2,44] Mic oo ganisms 2020,8, 1397 6 o 14 2.3. Cul i a ion, Ex ac ion, and Isola ion Fo cul i a ion, seed cul u es we e p epa ed by adding h ee mycelial plugs o ca. 0.25 mm 2 size each om well-g own yeas -mal aga pla es (YM 6.3 [ 45 ], supplemen ed wi h 20 g/L aga ) in o 250 mL shaking lasks con aining 100 mL o liquid YM 6.3 medium. Seed cul u es we e inocula ed on a o a y shake (In o s, Bo mingen, Swi ze land) o 13 d (23 ◦ C, 140 pm). Fo p epa a ion o he subme ged cul u es, 20 × 500 mL shaking lasks con aining 200 mL o ZM- 1 2 medium [ 45 ] we e inocula ed wi h 3 mL o seed cul u e. The lasks we e incuba ed on a o a y shake unde he a o emen ioned condi ions and consump ion o glucose was moni o ed by using glucose es s ipes (Mache ey-Nagel, Dü en, Ge many) daily. Besides glucose, a isual es ima ion o he ed colo o he cul u e b o h was aken in o accoun o de e mine he e mina ion o e men a ion. Acco dingly, h ee days a e he glucose es s ipes indica ed ha he glucose had been consumed (and he colo was o an in ense ed), he cul u es we e ha es ed and ex ac ed. A e e mina ion o e men a ions, supe na an and mycelium we e sepa a ed by cen i uga ion and subsequen gauze il a ion. The mycelium was ex ac ed wo imes, i s using 1 L o e hyl ace a e (E OAc) and hen 1 L o ace one as o ganic phases a 40 ◦ C in an ul a-sonic ba h o one hou . Bo h o ganic phases we e indi idually ex ac ed wice wi h ca. 1 L o deionized wa e using a sepa a o y unnel and he aqueous phases disca ded. The o ganic phases o he wo ex ac ions o he ZM- 1 2 medium we e combined and d ied in acuo o yield he c ude ex ac (526 mg). An aliquo o his ex ac was dissol ed in DMSO and subjec ed o HPLC-DAD/MS analysis (see Figu e S1). The c ude ex ac o he cul u e p epa ed in ZM- 1 2 medium was g adually sepa a ed using wo subsequen s eps o ch oma og aphy o yield he pu e compounds 1 and 2 . As a i s s ep, he c ude ex ac was dissol ed in 4 mL ACN:H 2 O (75:25) and ans e ed o an open solid phase ca idge (S a a ® X 33 µ m Polyme ic Re e sed Phase Tube, 1 g/12 mL, SN 8B-S100-JDG; Phenomenex, Ascha enbu g, Ge many). Elu ion was achie ed by using low acuum (ca. 800 mba ) and a s ep-g adien o H 2 O:ACN (90:10, 60:40, 30:70, 100:0; 4 mL pe s ep). The e luen s o he g adien s eps 90:10 and 60:40 we e combined acco ding o ESI-MS esul s and d ied in acuo, yielding 221 mg o ex ac . The esul ing c ude ex ac was u he sepa a ed by dissolu ion in ca. 5 mL o ACN:H 2 O (60:40) and subjec ion o a PLC 2250 HPLC sys em (Gilson, Middle on, WI, USA), u ilizing a Nucleodu ® C18ec column (125 × 40 mm, 7 µ m; Mache ey-Nagel, Dü en, Ge many) and sol en s: A: H 2 O+0.1% o mic acid, B: ACN+0.1% o mic acid, low: 45 mL × min −1 , ac iona ion: 15 mL, g adien : isoc a ic condi ions a 10% B o 10 min, ollowed by an inc ease o 70% B in 60 min, hen inc ease om 70% B o 100% B in 5 min, ollowed by isoc a ic condi ions o 100% B o 5 min. The ac ions we e combined acco ding o he DAD ch oma og am o he HPLC sys em: la iolin ( 1 ): R =29.5 − 31.0 min, 18.2 mg; 3,3 0 -bi la iolin ( 2 ): R =38.0 − 40.0 min, 19.2 mg. Ch oma og ams, DAD and MS aces o he pu e 1and 2a e depic ed in Figu e S2. Fu he mo e, o he g ow h media we e checked o p esence o he isola ed compounds 1 – 2 , as well as o he compounds o a chemo axonomic in e es . H. in adens was hus addi ionally g own in subme ged Q6- 1 2 , YM 6.3 (bo h [ 45 ]), MMK2 [ 46 ], and HLX [ 47 ], as well as he solid medium BRFT [ 45 ]. Fo his pu pose, cul u e condi ions as desc ibed abo e we e applied, wi h he di e ence o only one 500 mL shaking lask being incuba ed pe medium ( he solid BRFT medium was also no agi a ed du ing cul i a ion). Ex ac ion o mycelia was conduc ed as men ioned abo e, bu wi h only 200 mL o E OAc being used o ul a-sonica ion and he ace one ul a-sonica ion s ep omi ed. A e wa ds, he o ganic E OAc phase was ex ac ed wi h ca. 200 mL o wa e as desc ibed abo e. The supe na an , in u n, was di ec ly ex ac ed wi h ca. 200 mL o E OAc each. In he case o BRFT, no sepa a ion o supe na an and mycelium was possible due o he solid na u e o he medium, hence i was ea ed like he o he mycelia. HPLC-DAD/MS esul s o bo h supe na an s and mycelia a e depic ed in Figu e S3. 2.4. An imic obial Ac ici y and Cy o oxici y Assay Compounds 1 – 2 we e dissol ed in MeOH (1 mg/mL) o he bioac i i y assays. The sol en was also used as nega i e con ol. Mic oo ganisms 2020,8, 1397 7 o 14 Minimum inhibi o y concen a ions (MIC) we e de e mined in a se ial dilu ion assay as desc ibed p e iously [ 48 ]. Va ious es o ganisms o ungal and bac e ial o igin we e es ed o co e a b oad ange o mic oo ganisms: bac e ia: Bacillus sub ilis,S aphylococcus au eus,Mic ococcus lu eus,Ch omobac e ium iolaceum,Esche ichia coli, Pseudomonas ae uginosa; mycobac e ia: Mycolicibac e ium smegma is; ungi: Candida albicans,Schizosaccha omyces pombe,Muco hiemalis,Pichia anomala and Rhodo o ula glu inis. The MTT cy o oxici y assay was pe o med agains he cell lines L929 (mouse ib oblas s) and KB 3.1 (human papilloma i us- ela ed endoce ical adenoca cinoma) as desc ibed p e iously [49]. 2.5. Spec al Da a 2.5.1. Fla iolin ( 1 ): Red solid. MW =206.16 Da, MF =C 10 H 6 O 5 . NMR (ace one-d 6 , 1 H NMR: 500 MHz, 13 C NMR: 125 MHz): see Table S1; UV/ is (c =0.01 mg/mL, ACN): λmax ( ε )=214 (4.41), 262 (3.94), 301 (3.97) nm; ESI − MS: m/z206.92 [M+H] + , 204.79 [M − H] − ; HR − ESI − MS: m/z207.0284 [M+H] + (calcula ed o C10H7O5, 207.0288); R=4.8 min. 2.5.2. 3,30-Bi la iolin (2): Red solid. MW =410.29 Da, MF =C 20 H 10 O 10 . NMR (ace one-d 6 , 1 H NMR: 500 MHz, 13 C NMR: 125 MHz): see Table S1; UV/ is (c =0.01 mg/mL, ACN): λmax ( ε )=211 (4.56), 266 (4.39), 300 (4.15) nm; ESI − MS: m/z411.02 [M+H] + , 408.92 [M − H] − ; HR − ESI − MS: m/z411.0345 [M+H] + (calcula ed o C20H11O10, 411.0347); R=6.8 min. 3. Resul s 3.1. Molecula Phylogeny o H. in adens The newly gene a ed sequences o H. in adens and he da ase p esen ed in Figu e 1we e aligned as desc ibed abo e and cu a ed ia Gblocks, which lead o ou alignmen s spanning 347, 1113, 815 and 1047 posi ions (ITS, LSU, RPB2, and TUB2, espec i ely; c . Table S3). In o al, 1015 posi ions we e shown as pa simony in o ma i e, while 2049 posi ions ep esen ed cons an si es. The pa i ioned analysis (see Table S3 o a lis o selec ed bes - i models ollowing Bayesian-in o ma ion c i e ion, BIC) in e ed a ML- ee wi h a log-likelihood (lLn) sco e o − 40,100.7533 (Figu e 1). The ee was oo ed o he ou g oup consis ing o ep esen a i es o he G aphos oma aceae and Xyla iaceae (O) wi h he Hypoxylaceae ecei ing maximum BS and 99% SH-aLRT suppo , wi h H. papilla um appea ing basal o all o he clades (Hx1). The Hypoxylaceae u he mo e show a pa aphyly o Hypoxylon, spli in o one s ongly suppo ed clade (Hx2, 98% SH-aLRT /95% BS) and one s ongly suppo ed sis e clade (78% BS, Hx7) (85%/56%) pu nex o Annulohypoxylon (A, 100%) and Jack oge sella (J, 98%/100%). Fu he mo e, one non-suppo ed clade con aining H. agi o me and allies (Hx3) was placed as sis e g oup nex o a clade con aining mos ly o he gene a like Py enopolypo us (P, 100% suppo ), Daldinia and allies (99%/100%; E, D, R, T), Hypomon agnella (Hy, 100%) and o he nes ed sequences wi h non- o low suppo ed posi ions o Hypoxylon and En onaema (E, Hx4, Hx5, Hx6) wi h mode a e BS and high likelihood- a io suppo (95%/58%). These pa e ns we e al eady desc ibed by Wend e al. [ 2 ], Si e al. [ 20 ], and Pou moghaddam e al. [ 39 ]. The H. in adens-de i ed sequences we e placed nex o H. ugodes wi h maximum suppo (Hx5). Mic oo ganisms 2020,8, 1397 8 o 14 Figu e 1. In e ed maximum-likelihood (lLn = − 40,100.7533) ee o a molecula phylogeny o sequenced loci o ibosomal o igin (ITS, LSU) and p o einogenic genes (RPB2, TUB2), wi h pa i ion-wise assigned e olu iona y models. SH-aLRT (>85%) and BS (>50%) suppo is gi en a i s and second posi ion a espec i e b anches. The labeled clades co espond o he gene a Annulohypoxylon (A), Daldinia (D), En onaema (E), Hypoxylon (Hx 1 − 7), Hypomon agnella (Hy), Jack oge sella (J), Py enopolypo us (P), Rhopalos oma (R), Thamnomyces (T) and he oo ed ou g oup (O). The clades ha e been highligh ed by al e na i e g ey and ligh blue backg ound o allow o easie dis inc ion. The posi ion o H. in adens is highligh ed in bold. No e: The b anch leng h o H. lienhwacheense has been unca ed, as indica ed. B anch leng h equals subs i u ions pe si e. 3.2. S uc u e Elucida ion o Fla iolin ( 1 ) and 3,3 0 -Bi la iolin ( 2 ) and Analysis o Ex ac s om o he Cul u e Media Two seconda y me aboli es we e isola ed om an ex ac (c . Figu e S1) o cul u es o H. in adens in ZM- 1 2 medium, which we e ul ima ely iden i ied as he known naph hoquinones la iolin ( 1 ) and i s dime , 3,3 0 -bi la iolin ( 2 ) (see Figu e 2). A combina ion o 1D and 2D NMR, as well as a di ec compa ison o UV/ is spec al da a wi h e e ence da a [50] was applied. Mic oo ganisms 2020,8, 1397 9 o 14 Figu e 2. Chemical s uc u es o la iolin ( 1 ) and 3,3 0 -bi la iolin ( 2 ), seconda y me aboli es isola ed om cul u es o Hypoxylon in adens. Fla iolin ( 1 ) was isola ed as a ed solid and shown o possess a molecula o mula (MF) o C 10 H 6 O 5 by HR-ESI-MS. Analysis o 1D and 2D NMR da a led o he s uc u e p oposal o 1 . The comple e s uc u e could be con i med when di e en long ange coupling cons an s in 1 H/ 13 C he e onuclea mul iple-bond co ela ion spec oscopy ( 1 H/ 13 C HMBC) expe imen s (CNST 13 =2 Hz, 6 Hz) we e used, which allowed o 3 J, as well as 4 Jco ela ions o be obse ed. Taken oge he , hese co ela ions allowed he con i ma ion o he isola ed compound as la iolin (1). Fo he ed solid 3,3 0 -bi la iolin ( 2 ), a MF C 20 H 10 O 10 was calcula ed. I s s uc u e was con i med ia NMR spec oscopy o be a symme ical dime o 1 . This was deduced by i s molecula o mula, which ep esen ed a duplica ion o a oms compa ed o 1 wi h a o mal loss o wo hyd ogens, highly simila UV/ is spec a (Figu e S2), and he simila i y o p o on and ca bon signals in NMR da a o 1 – 2 . The b idging a om in 3,3 0 -bi la iolin was de i ed o be C-3/C-3 0 , as he 1 H NMR signals o 3-H we e missing in 2as compa ed o 1. NMR da a, key co ela ions, as well as 1D and 2D spec a can be ound in Table S1, Figu e S4 and Figu es S5–S16, espec i ely. The HPLC-DAD/MS ch oma og ams de i ed om i e sc eening cul u es in six di e en media in which H. in adens had been e men ed we e checked o he p esence o compounds 1 and 2 , as well o he chemo axonomically ele an seconda y me aboli es. Aside om he ZM- 1 2 medium, whe e 1 and 2 we e majo componen s, we we e only able o de ec hem in HLX medium (c . Figu e S3B). No ably, compounds 1 and 2 we e no ound in ex ac s om he YM 6.3 medium, which was he s anda d medium in a comp ehensi e chemo axonomic s udy by Bi ze e al. [ 35 ] (Figu e S3C). Fu he mo e, hey we e nei he p esen in he ex ac s om subme ged MMK2 and Q6- 1 2 media, no in he solid, ice-con aining BRFT medium (Figu e S3D–F). No known compounds om ela ed species (including mellein de i a i es, which we e p esen in ex ac s om subme ged cul u es in YM medium o almos all Hypoxylon sp. p e iously s udied [ 35 ], could be assigned o he la ge numbe o peaks p esen in he ex ac s. 3.3. An imic obial and Cy o oxic Ac i i ies o Fla iolin (1) and 3,30-Bi la iolin (2) The an imic obial and cy o oxic ac i i ies o la iolin ( 1 ) and 3,3 0 -bi la iolin ( 2 ) we e e alua ed as desc ibed in he me hods sec ion and a e summa ized in Table S2. An ibac e ial ac i i y o 1 agains S aphylococcus au eus wi h a minimum inhibi o y concen a ion (MIC) o 66.7 µ g × mL −1 was de e mined, which was e y weak in compa ison o he e e ence oxy e acycline (MIC =0.4 µ g × mL −1 ). No ac i i y o 1 – 2 agains any o he o he mic obial es o ganisms was obse ed. E alua ion o he cy o oxic e ec s o 1 – 2 on mammalian cell lines esul ed in 1 exhibi ing a hal -maximal inhibi o y concen a ion (IC 50 ) o 92.2 µ M agains L929 mouse ib oblas s. The e e ence epo hilone B, in u n, exhibi ed an IC 50 o 0.00006 µ M agains L929. O he cy o oxic e ec s we e no obse ed agains he chosen cell lines.