Amer. J. Bot. 63(6): 838-844. 1976.

STRUCTURE AND DEVELOPMENT OF CLEISTOIODOPHANUS CONGLUTINATUS GEN. & SP. N. (ASCOBOLACEAE)l

JOSE L. BEZERRA2 AND JAMES W. KIMBROUGH Departmentof Botany,University of Florida, Gainesville32611

A B S T R A C T Cleistoiodophanusrepresents a new coprophilousgenus of the tribe Iodophaneae in the Ascobolaceae (). The only speciesthus far discovered,C. conglutinatus,is described and illustrated.Aspects of its cytologicaldevelopment are describedfrom cultures obtained fromapothecia found on sheep dung near Gainesville,Florida. Plasmogamyoccurs in aco- gonialcoils, two or threecells of whichgive riseto ascogenoushyphae. Ascogoniaare quickly enclosedby vegetativehyphae and the ascocarp continuesin a cleistohymenialdevelopment. UnlikeIodophanus and relatedgenera, the excipulumremains intact even afterspore matura- tion and the asci push throughthe epihymenialregions to release spores. The asci are charac- teristicallythickened at theirapices, diffuselyamyloid, and somewhatsaccate. The asci are predominantly8-spored, but have been foundwith four or 16 sporesper ascus. A previously undescribedOedocephalum imperfect stage was inducedin culture.

IN A SEARCH for coprophilous discomycetesin manentia, albo colore praedita; excipula ectalis Gainesville,Florida, a funguswith lightcolored, cyanophilica,cellulas rectangularesvel quadratas roundishapothecia was foundto formabundantly habens; excipulamedularis cyanophilica, cum tex- on sheep dung when placed in a moistchamber. tura globulosa; asci generaliteroctospore, diffuse Cultures exhibitingan Oedocephalum imperfect amiloides,large clavati, cum parietibuscrassis in stage were easily establishedfrom fragmentsof juventute,pedicelati; ascospori uninucleati,hy- apotheciatransferred to agar media. Microscopic alini, ellipsoides,laeves, perisporiummucilagino- examinationof squash mounts and cryostatsec- sum ostentantes;paraphyses ramosae, hyalinae, tions of apothecia revealed a typicaldiscomycete septatae,multinucleatae. hymenium,but the asci remainedcovered by the Momentumconidiale: Oedocephalum Preuss. excipulumeven when fullydeveloped. The color Species typus: C. conglutinatussp. n. and externalmorphology of the apothecium,the Derivation: Greek, kleistos= closed, plus texturaglobulosa of the medullaryexcipulum, the lodophanus,a closelyrelated . natureof the asexual stage togetherwith the dif- Apothecia gregariousto cespitose, developing fuselyamyloid reaction of the asci, and the large, cleistohymenially,remaining closed, lightcolored; ellipsoidal and hyaline ascospores related the ectal excipulumof texturaprismatica, cyanophilic, fungusvery closely to lodophanus. However,the of rectangularor quadraticcells; medullaryexci- cleistohymenialascoma remaining permanently pulumcyanophilic, of texturaglobulosa; asci usu- closed, the saccate, stalkedand thick-walledasci, ally 8-spored,diffusely amyloid, broadly clavate, the smooth ascospores (present only in I. dif- thick-walledin youth,stalked; ascospores uninu- formis), and the differenttype of ascogonia cleate, hyaline,ellipsoid, smooth, a mucilaginous eliminatedlodophanus as a genusto accommodate perisporepresent; paraphyses branched, hyaline, the present species. Thus a new genus is pro- septate,multinucleate; conidial stage: An Oedo- posed for this unique Discomycete: cephalumPreuss. Coprophilous. Cleistoiodophanusgen. n. Cleistoiodophanusconglutinatus sp. n. (Fig. 1-32). Apothecia coprophila, gregaria vel cespitosa, sese cleistohimenialiterevolventia, occulsa, autem, Apothecia gregariavel cespitosa, sese cleisto- himenialiterevolventia, primo globosa, deinde 1Received for publication25 June 1975. irregulariterlobato-sulcata, occlusa, autem,man- 2Permanent address: Departmentof Mycology,Uni- entia,_colorecarneo vel albo-flavopraedita, ob- versidadeFederal de Pemambuco,Recife, PE, Brazil. scuriorapost maturitatem,leviter tomentosa, 0.5- The authorswish to acknowledgethe assistanceof Dr. ectalis, 20-35 tm crassa, F. B. Leal (UniversidadeFederal de Pemambuco,Bra- 3.0 mm diam; excipula zil) in supplyingthe Latin diagnosis,and that of Dr. cyanophilica,cellulas habens protensas, in 2-4 MeredithBlackwell and Dr. Gerald Bennyfor darkroom straturis,rectangulares vel quadratas, angulatas, assistance. x 6.0-10.5 jum,cum tex- Florida AgriculturalExperiment Station Journal Series multinucleatas,6.0-22.0 No. 5955. turaprismatica, haec cellulae suntlateraliter com- 838 July, 1976] BEZERRA AND KIMBROUGH-CLEISTOIODOPHANUS CONGLUTINATUS 839 pressae et externe hyphis hyalinis circumdatae, 21 x 11-16 jm; denticlesminute, sometimes in- quae apotheciis aspectum tomentosumofferunt; conspicuous,varying in numberfrom 5-16, 1 jum excipula medulariscyanophilica, cum texturaglo- long; conidia subglobose to ellipsoid, botryose, bulosa et cellulisellipsoidibus, 15.0-27.0 x 13.5- solitary,slightly verrucose, 8.5-13.5 (-15.0) x 15.0 tm;asci octospori,aliquando 4 vel 16 sporos 6.0-9.0,um,formed as blown-outends of the apex habentes,diffuse amiloides, large clavati,in apice of the denticles,germinating readily by 1-2 germs rotundi,cum parietibuscrassis in juventute,ped- tubes. icelati, (83.0-) 90.0-120.0 x 25.0-30.0 (-37.5) jum; ascospori uninucleati,distichi vel tristichi, Derivation: Latin,conglutinatus = unitedfirmly hyalini,large ellipsoides,in polis rotundi,laeves, together,referring to the arrangementof the ad maturitatemparietes tenues ostentates,15.0- apothecia. 19.0 x 9.0-13.5 tum,cum perisporiomucilaginoso; paraphyses hyalinae, ramosae, septatae, multi- Holotype: On sheep dung,Veterinary Science nucleatae,leviter in apice dilatatae,3-6 Km diam. Farm, Universityof Florida Campus, Gainesville, Momentumconidiale: Oedocephalum Preuss: Alachua County, Florida, Jan. 18, 1973, J. L. conidiophorisese ab hyphisrepentibus elevantes, Bezerra (FLAS-49579). tanquamrami laterales, erecti, simplices, cylindrici, septati, hyalini, 37.0-135.0 x 4.5-7.5 m; septi, MATERIALS AND METHODS-Culture and devel- 2-5, non constricti,primus in conidiophoribasi opment-Pellets of sheep dung were collectedon formatuset postremusin basi vesiculae; vesicula January18, 1973, at theVeterinary Science Farm, terminalis,aliquando intercalaris,pyriformis, den- Universityof Florida Campus, Gainesville,Flor- ticulata,17-21 x 11-16 jm; denticuliminuti, ali- ida. The materialwas placed in a moistchamber quando inconspicui,5-16, 1 Kmlong; conidia sub- at room temperatureand apothecia of the globosa vel ellipsoidia,botryosa, singularia, leviter were detectedsix days afterincubation (Fig. 16). verrucosa, 8.5-13.5 (-15.0) x 6.0-9.0 m, ut Squash mountsand cryostatsections were made apicum denticulorumdilatationes formata, 1-2 of the apotheciaand examineddirectly in wateror tubisgerminativis propere germinantia. stainedin cottonblue (Korf, 1973), 5 % aqueous In arietis faecibus. Typus: J. L. Bezerra, Congo red (Kimbrough,1969), and Melzer's Re- Gainesville, Florida, America septentrionalis agent (Korf, 1973). Nuclear stainingwas done (FLAS F-49579). by the HCl-Giemsa methodas describedby Fur- Apothecia gregariousto cespitose, developing tado (1968) and by the aniline-bluemethod of cleistohymenially,globose at first,becoming ir- Tu & Kimbrough(1973). Cultureswere estab- regularlylobulate-sulcate but remainingclosed, lished by washing some apothecia in steriledis- flesh-coloredto light yellow, darker on drying, tilled water and placing apothecial fragmentson finelytomentose, 0.5-3.0 mm in diam; ectal exci- agar media. Aftercompletion of the studies,the pulum 20-35 Kmthick, cyanophilic, of 2-4 layers dung pelletsbearing apothecia were driedat 60 C of elongate, rectangularor quadratic, angular, and accessionedto the Universityof Florida My- multinucleatecells, 6.0-22.0 x 6.0-10.5 pm, a cologicalHerbarium. texturaprismatica, laterally compressed and ex- ternallycovered by hyalinehyphae giving a tomen- OBSERVATIONS-Cultural characteristics-The tose appearance to the apothecia; medullaryex- fungusdevelops slowly and formsscanty myce- cipulum cyanophilic, of textura globulosa, of lium on mycologicalagar (MA), potato-dextrose- globose to ellipsoid cells, 15.0-27.0 x 13.5-15.0 agar (PDA), cornmeal-malt-yeastextract-agar jum;asci 8-spored,sometimes 4 or 16-spored,dif- (CMMY), V-8 agar, V-8 agar plus dung extract, fusedlyamyloid, broadly clavate, rounded above, and dung extract-oatmeal-agar(DOA). Conidia thick-walledin youth, stalked below, (83.0-) are formedabundantly on all testingmedia but 90.0-120.0 x 25.0-30.0 (-37.5) ym; ascospores only sexual initials,and immatureapothecia are uninucleate,bi- or tri-seriate,hyaline, broadly el- produced. lipsoid,rounded at the tips,smooth, thin-walled at maturity,15.0-19.5 x 9.0-13.5 Am, a mucilagi- Mycelium-The vegetativehyphae are consis- nous perispore present; paraphyses hyaline, tently branched and multinucleate. Woronin branched,septate, multinucleate, slightly inflated bodies are observednear the septa. at thetip, 3-6 jumin diam. Conidial stage, an OedocephalumPreuss: co- Conidiophoresand conidia-The conidiophores nidiophoresarising as lateral branches fromre- arise as small protuberancesfrom the repenthy- pent hyphae, erect, simple, cylindrical,septate phae. After a period of elongation,an apical hyaline,37.0-135.0 x 4.5-7.5 m; septa in num- swelling develops at the tip of the developing ber of 2-5, non-constricted,the firstone formed conidiophoreswhich gives rise to the ampulla at thebase of theyoung conidiophore and the last (Fig. 3). Thornlikedenticles then form over the one at thebase of the ampulla; ampullaeterminal, ampulla (Fig. 3). Conidial formationis initiated sometimesintercalary, pyriform, denticulate, 17- by apical swellingof the denticles(Fig. 2, 3) and 840 AMERICAN JOURNALOF BOTANY [Vol. 63

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- Fi. 5.Clisoidohauscogltiats.1.Maur cniiaatacedt an emp ityapl.x , 2 3 Coidohoe an: bern amulawt dvlpigcoii. ,00 . aue oiiu it ogheipoe 1,000 5.Trecndathmideoewh a ger tb.X 0. 6,Mliulae an infate veeatv July, 1976] BEZERRA AND KIMBROUGH-CLEISTOIODOPHANUS CONGLUTINATUS 841 continueswith the migrationof cytoplasmand otic divisiontakes place and a meioticspindle is nucleiinto the developingconidia. When matura- formed,generally parallel to the long axis of the tionis completed,the conidia become surrounded ascus (Fig. 19, 20). These meioticprocesses re- by a thick,slightly verrucose outer wall (Fig. 4), sult in two verticallyarranged nuclei (Fig. 21). and the ampulla and denticlesappear empty(Fig. The second divisionis synchronousand the suc- 1). Mature conidia are arrangedin a clustered ceedingfour nuclei occupy the middle of the ascus fashionaround the ampulla (Fig. 1) . Justbefore (Fig. 22). Mitotic divisions of the tetrad are germination,the conidia swell and formusually synchronous(Fig. 23). The nucleolusreappears one, sometimestwo, protrusionswhich elongate aftereach division (Fig. 22). Peculiaritiesdur- intogerm tubes (Fig. 5). ing spore formationsometimes lead to the incor- porationof two nuclei into one spore,resulting in Ascogonium-The ascogoniumarises by swell- four-sporedasci (Fig. 24). Occasionallyan addi- ing and beadingof vegetativehyphae in whichthe tional mitoticdivision gives rise to the formation cytoplasm becomes condensed (Fig. 6). The of sixteen spores inside one ascus (Fig. 25). ascogonialcells become morerounded at the time Ascal tips are initiallythick-walled (Fig. 26), but ensheathinghyphae startto encirclethe ascogo- become thin with inconspicuousareas of dehi- nium. The ensheathinghyphae divide and pro- sence at maturity(Fig. 27). duce a mass of cells aroundthe ascogonialinitials (Fig. 7, 8). No antheridiaor male elements Spores-Each ascus usually forms eight uni- were observedfusing with the ascogonium. Usu- nucleate ascospores (Fig. 30). The ascospores ally two or threeascogonial cells can be observed are somewhatthick-walled when young (Fig. 29), withinthe mass of cells (Fig. 7, 8). Ascogenous but become thin-walledand remain smooth at hyphaebegin to grow out fromthe multinucleate maturity.The outer perisporiclayer is mucilagi- ascogonia. The ascogenoushyphae start to branch nous and may be slightlywrinkled in the mature and formcroziers at theirends (Fig. 10, 11). spore (Fig. 28). Paraphyses-The paraphyses appear in very Ascocarp development-The developmentof early stages of ascal development,are multinu- the apotheciais cleistohymenial.The mass of cells cleate, septate,branched, hyaline, and arise from around the ascogonial initialsbecomes the archi- cells whichsupport the croziers (Fig. 31, 32). carp (Fig. 9, 14). As the archicarpincreases in size and the ascogenoussystem grows, an excipu- DISCUSSION-Cleistoiodophanuspossesses the lum develops which is made up of multinucleate mostimportant features attributed to the Ascobo- subglobosecells (Fig. 12). The externalcells of laceae by Korf (1973): ascospores very thick- the excipulumbecome elongated,angular to quad- walled when young, egguttulate,uniformly uni- ratic (Fig. 13), and those of the basal excipulum nucleate,hyaline and asci diffuselyblue in iodine. remainsubglobose. Multinucleate,branched, fil- The cleistohymenialdevelopment of Cleistoiodo- amentous paraphysesoutgrow the asci and are phanusis matchedonly by lodophanus, foundlying between the top of the asci and the and , among the Ascobolaceae. How- excipulum (Fig. 17). Frozen sections showed ever, while Cleistoiodophanusapothecia always that the excipulum encloses the asci and para- remainsclosed, those of the othergenera open at physes even aftersporogenesis is completed;the maturity. ascocarp will not separate to expose the hy- Ascogonialontogeny in C. conglutinatusis very menium but small fractureswill form through similar to that of Iodophanus granulipolaris whichthe ripeningasci push to liberatethe asco- (Milam, 1971); however,it differsin some as- spores (Fig. 15). Apothecia remainglobose and pects. The ascogenoushyphae are of the pleuror- tightlyclustered (Fig. 16). hynque type (formingcroziers) similar to the speciesmentioned above and to Lasiolobus ciliatus Asci-The two haploid nuclei of the penulti- (Conway, 1975). As in I. granulipolaris,the mate cell of the crozierfuse togethergiving rise ascogonial coil elongates and yields "privileged" to the ascus mothercell witha prominentdiploid cells that give rise to ascogenous hyphae which nucleuscontaining one or two nucleoli (Fig. 18). become surroundedby investinghyphae. The As the youngascus increasesin size the firstmei- ascogonial coil does not fuse withmale elements.

roundedby investinghyphae. X 500. 9. Archicarp.X 250. 10. Crozier with a dikaryoticpenultimate cell (ar- row) priorto karyogamy.X 1,000. 11. Croziers,one witha diploidpenultimate cell (arrow) afterkaryogamy. X 1,000. 12. Multinucleate,rounded ectal cells of the excipulumin a squash mount. X 1,000. 13. Longitudinal sectionthrough periphery of the ascocarpshowing empty asci, and paraphysescovered by quadraticectal cells of the excipulum. X 1,000. 14. Longitudinalsection through an archicarpwith undifferentiated asci (arrows). X 250. 15. Cross sectionof an ascocarp showingmechanism of ascosporedehiscence through breaks in the excipulum(ar- rowheads). X 100. 842 AMERICAN JOURNAL OF BOTANY [Vol. 63

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yses (P), ascal tips (A), and excipularcells (E). Note paraphysealapices lyingbetween ascal tips and excipular cells. X 1,000. 18. Young ascus containinga large diploidnucleus with two nucleoli. X 1,000. 19, 20. Firstmei- otic division. Note spindlesoriented vertically or obliquelyin relationto longitudinalaxis of ascus. X 1,000. 21. Two nucleiresulting from the firstmeiotic division. X 1,000. 22. Threenuclei resulting from the secondmeiotic di- vision (the fourthnucleus is out of focusand is not shown). Note the reappearanceof the nucleolus(arrow). X 1,000. 23. Five nuclei resultingfrom the mitoticdivision after meiosis (the threeother nuclei are out of focusand notshown'. X 1,000. July 1976] BEZERRA AND KIMBROIJGH-CLEISTOIODOPHANUS CONGLUTINATUS 843

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The trichogyne-likestructures seen in lodophanus all descriptionsof Oedocephalumreported in the granulipolariswere not observedin C. conglutina- literaturebecause we believed that it was super- tus. Crozier formation,ascus development,and fluousto describeit as a separatetaxon. ascosporogenesisin C. conglutinactus,to the extent thatthey were studiedin thiswork, did not differ LITERATURE CITED significantlyfrom I. granuulipolaris.The n,uclear conditionof the excipularcells and paraphysesis CONWAY, K. E. 1975. The ontogenyof Lasiobolus ciliatus (Pezizales, Ascomycetes). Mycologia 67: also identical in both species. Despite thp fact 253-263. that the apothecia of Cleistoiodophanusdo not FURTADO,J. S. 1968. Basidial cytologyof Exidia nu- open at maturity,the apothecial developmentis cleata. Mycologia60: 9-15. the same as in lodophanus. Therefore,we think GAMUNDI, I. J., AND M. E. RANALLI. 1964. Estudio thatboth genera should be placed in theAscobola- sistematicoy biologico de las Ascobolaceas de Ar- ceae. gentina. I. Nova Hedwigia 7: 517-533. The developmentof conidiophoresand conidia JENG, R. S. 1971. Studieson the morphologyand de- in C. conglutinatusis basically the same as in velopmentof lodophanuscarneus (Pers. per Pers.) lodophanus carneus (Pers. per Pers.) Korf, in Korf. In Kimbrough& Korf. M.S. Thesis, Uni- Kimbrough& Korf,as reportedby Jeng (1971). versityof Toronto,Toronto, Canada. The conidialmeasurements, however, differ for I. KIMBROUGH, J. W. 1969. NorthAmerican species of carneus and for lodocephalum glomerulosum (Pezizeae, Pezizaceae). Mycologia 61: (Bull. ex Harz.) Sacc. Iodophanus carneus has 99-114. small, globose, smooth (?), 5.6-7.0Ojm diam KORF, R. 1973. Discomycetesand Tuberales,p. 249- conidia (Jeng, 1971) and 0. glomerulosumhas 319. In G. C. Ainsworth,F. K. Sparrowand A. S. Sussman[edi], The fungi:an advancedtreatise, Vol. large, ovoid, smooth, 20-27 (-28) X 8.3-13.5 IV A. AcademicPress, New York and London. conidia (Gamundi and Ranalli, 1964). Jeng tm MILAM, J. R. 1971. Cytologicaldevelopment of lodo- (1971) referredto "smooth conidia" in her de- phanus granulipolaris(Pezizazeae, Ascomycetes). scriptionof I. carneus despite the fact that she M.S. Thesis, Universityof Florida, Gainesville, presentedan electronmicroscope photograph of Florida. a conidiumwith warts on the wall which were Tu, C. C., AND J. W. KIMBROUGH. 1973. A rapid noted and labelled. We did not attemptto com- stainingtechnique for Rhizoctonia solani and related pare the conidial stage of C. conglutinatuswith fungi.Mycologia 65: 941-944.