Developmental studies in Actinospora and three similar aquatic hyphomycetes

Trans. Br , mycol. Soc. 67 (2) 207-222 (1976) Printed in Great Britain

also identified as Actinospora megalospora, whichproduced a dark colony on Goos ' corn-meal agar(GCMA) and sporulated readily without immer-sion. Its less complex conidiophores proliferatedpercurrently, leaving successive annellate scars attheir apices. Fertile pycnidium-like bodies werealso produced. No formal description was pro-vided .

In view of this situation A. megalospora was re-isolated from a stream in Devon (England) andinduced to sporulate under conditions describedbelow. The resultant structures match the typematerial and have also been authenticated byIngold (pers. comm. ). Ontogenic studies on thisisolate necessitate an emendation of this stillmonotypic genus, which can be safely recordedonly from England.

As already remarked by Ingold (1952), A .megalospora does not sporulate on 2 % malt extractagar or other common media; neither does it fruitreadil y on GCMA or rabbit food decoction agar(Goos, 1970). When grown on GCMA and sub-merged in forcibly aerated but unchanged steriledistilled water a few mis-shapen conidia appear inscattered patches. However, when a small piece ofagar culture is placed in a slide chamber with cleansterile distilled water flowing at a constant ratethrough it (Fig. 1), conidiophores differentiateafter a few hours, and abundant conidia, highlyhom ogeneous in size and shape, are produced insuccessive crops. Numbers are highest in the firstflush, but even after several days conidia stilldevelop normally on conidiophores growing fromthe vegetative hyphae or arising percurrently fromthe ends of the spent dichotomies (Figs 2 C, 3).Also interesting is the extreme sensitivity ofconidial development to the presence of bacteria.The sporulating chamber, therefore must be keptsterile. A slide tissue culture chamber (Sterilinproducts) can also be adapted as a constant-flowchamber.

DEVELOPMENTAL STUDIES IN ACTINOSPORA AND THREE

SIMILAR AQUATIC HYPHOMYCETES

By E. DESCALS c., A. NAWAWI* AND J. WEBSTER

Department of Biological Sciences, University of Exeter

On the basis of ontogenetic studies of pure cultures submerged in a constant flow of water,Actinospora megalospora Ingold is redescribed and emended, and three other new fungi,Porocladium aquaticum Descals, Brachiosphaera tropicalis Nawawi and Clavariana aquaticaNawawi are described and illustrated. The new combination Brachiosphaera jamaicensis(Crane & Dumont) Nawawi is proposed.

Ingold (1952) described Actinospora megalosporadeveloping on bark of a submerged twig ofCrataegus in England. Its conidia had severalbroad-based arms radiating simultaneously froma globose to subglobose central body whichdeveloped from swelling of the terminal cells ondichotomously branched conidiophores. Single-conidium isolates produced a rather scanty,deeply immersed, transparent mycelium whichdid not sporulate even when submerged in water.

In over 20 papers, its world-wide occurrencehas been claimed on the basis of identifications ofdetached conidia collected from foam or scum. Ithas been recorded from Nigeria (Ingold, 1956,1959; Alasoadura, 1968), Ghana (Dixon, 1959),Uganda (Ingold, 1958), Jamaica (Hudson & Ingold,1960), Sweden (Nilsson, 1962, 1964), NewZealand (Tubaki, 1965 a), East New Guinea(Tubaki, 1965b), United States (Crane, 1968 ;Conway, 1969), Ukraine (Dudka, 1970), U .S.S.R.(Arnold, 1970; Dudka, 1970), Hungary (Gonczol,1971), Swaziland (Ingold, 1973), Romania (Toth,1973), Malaysia (Nawawi, 1973), Canada (Baer-locher & Kendrick, 1974) and again in England(Jones, 1965; Iqbal & Web ster, 1973). An artificialkey for its identification is also provided by vonArx (1970), but the key omits criteria based onontogeny, which are fundamental for its segrega-tion from other genera with tetraradiate conidia.

Conflicting records first appeared in a mono-graph on the aquatic hyphomycetes by Petersen(1962 ) where Actinospora was keyed out as havingconidial arms developing in succession. Conway(1970, Fig . 1A) illustrated a conidium from NewYork State labelled A. megalospora, with arms upto three times those of the type. However, thedimensions of the arms were given as 100-180lIm x 8 I'm which were tho se of Ingold (1952).Goos (1970) isolated a hyphomycete from Hawaii,

* Present address: Department of Botany, Univer-sity of Malaya, Kuala Lumpur.

e bed f a

208

2cm

Actinospora and related fungi

Septa over the entire conidiophore, includingthose separating future conidia, were laid downprior to differentiation of the apical cells. But itwas quite evident, even at zoo x magnification,that the dark-yellow, foamy protoplasmic contentscould push through the conspicuous septal poresin intermittent thrusts, filling up the main bodiesof the conidia during their development. Cyto-plasmic continuity exists throughout the entiresporulating structure, and the significance of theterms thalloconidium and aleurioconidium, usedin describing this fungus, is questioned.

The taxonomic dimensions on which the re-description of this species is based are generallysmaller than those ofmaterial found in nature (Fig.3 F) . The degree of conidial branching too isreduced. This probably has to do with the environ-mental conditions under which pure cultures areinduced to sporulate.

The description below is based on specimenExeter 3z05. The dimensions from the originaldescription are in parentheses.

Fig. 1. Details of the constant-flowslide chamber. A,Perspex slide; B, Agar plug cut in half, sporulating onthe free surface; C, Sporulating chamber; D, Holesbored through the perspex slide, connecting thechamber with the PVC tubes, one of which supplieswater and the other acts as a drain j E, Coverslips onboth sides of the chamber, allowing for microscopicobservation from either side; F, The chamber is sealedwith hot paraffin wax applied with a Pasteur pipettealong the sides of both coverslips.

Several sequences of sporulation were followedunder the microscope and illustrated. Time-lapsecinematography was also carried out for all fourfungi described here (Webster, Nawawi & Descals,1975)·

In every case conidiophores of A. megalosporabehaved in the same way with all apical cells of thedichotomies simultaneously differentiating intoconidia. About Z4 h later cells that had beensupporting the terminal conidia would swell up,again simultaneously, and produce four to fiveequally arranged arms. Conidia produced initiallywould gradually slide over the surface of theunderlying ones and remain in situ forming atangled mass. Eventually the flowing water wouldcarry the conidia away. Sometimes conidiophorebranches were three-celled, each successivelydeveloping into a conidium. Upon conclusion ofsporulation only short forks would remain on thedichotomies. Although less complex heads havebeen illustrated here for easier observation,conidiophores supporting many more conidia werecommon.

ACTINOSPORA Ingold emend. Descals Trans. Br.mycol. Soc. 35: 66 (195z).

Saprophytic hyphomycete, Conidiophores hya-line, acroauxic, macronematous, mononematous,solitary, straight, unbranched or more commonlybranching dichotomously once or several times.The cells of the dichotomies develop basipetallyinto conidia which consist of subglobose toopyriform main bodies with typically four ormore arms radiating simultaneously.

ACTINOSPORA MEGALOSPORA Ingold Trans. Br.myco!. Soc. 35 : 66 (195Z). (Figs. zA-C, 3)

Submerged aquatic fungus. Pure culture:grown on Goos corn-meal agar at 15 °C underdiffuse light. Colony growth moderately fast,attaining 9 ern in 60 days. Aerial mycelium absent.Immersed hyphae sparsely branched, septate, withhyaline walls; protoplasm turning dark-yellowwith age; conidiophores hyaline-walled, with darkyellow protoplasm; acroauxic, macronematous,mononematous, solitary, straight, simple or morecommonly with a basal stipe 100-300 11m long x8-10 11m broad, and an apical head branchingdichotomously once or several times, the terminalportions o-z septate, 100-ZOO 11m long x 8-1zl1mwide, initially of uniform width, but later develop-ing basipetally into conidia consisting of a swollenmain body and 4-5-{8) straight arms, arisingsimultaneously; one arm always distal; the othersarranged equidistantly around the base andradiating slightly backwards. Mature detachedconidia consisting of a subglobose to obpyriform

Fig. 2. (A-C). Actinospora megalospora. (A-B). Successive stages in conidial development, from pure culture (Exeter No. 3205)(Scale A). (C). Conidiophore showing per current growth and the differentiation ofa second dichotomy (Scale B). (D-G). Porocladiumaquaticum. (D-F). Successive stages in conidial development and proliferation, from pure culture (Exeter No. 3206 ) (Scale C) . (G ).Detail of tretic origin of a conidium (same culture) (Scale C).

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210 Actinospora and related fungi

Fig. 3. Actinospora megalospora Ingold. A-E from pure culture. The numbers indicate the sequence ofsporulation. A, Dichotomy prior to differentiation; B, Arms appearing out of the terminal conidia; C,Terminal conidia fully developed and detached. Subterminal conidia start developing their arms; D,Later stage; E, Some conidia have already floated away. One conidium is 5-armed (No.2); F, Detachedconidium collected from foam in the R. Culvery, Devon (VII. 1975).

E. Descals and others 211

main body, 90,u120 ,um long and 45-65(35-4°) ,umbroad at the widest point, with dark-yellow proto-plasm. The arms are straight, 100-190,um (100-180,um) long and 12'5-20 ,um (8) ,um wide at thebase, broadly based, and tapering graduallytowards a more or less distinct clavate apex;protoplasm hyaline. Conidia released successivelyafter full development. Germination on agartypically from the arms. Perfect state unknown.Collection: single conidium from foam on the R.Teign, Devon, England, Jan. 1974. Type speci-men: deposited at Herb. IMI 47684 (bark ofCrataegus sp. with two conidiophores bearingseveral conidia; mounted in lactic acid). Authenti-cated material deposited at Herb. IMI 199903 andHerb. University of Exeter No. 3205.

POROCLADIUM Descals gen. nov.

Hyphomyces saprophyticus. Conidiophora hyalina,acroauxica, macronematosa, mononematosa, caespitosavel solitaria, primum recta, mox autem geniculata, sineramis. Cellulae conidiogenae treticae, in conidiophorisincorporatae, terminales sunt vel intercalares fiunt,cylindricae, prolificantes saepius sympodiali modo velnonnumquam determinatae. Tretoconidia solitaria,acrogena, hyalina, staurospora. Brachia septata, simulradiantia e corpore principali tumido,

Species typica: Porocladium aquaticum Descals n.sp.

Saprophytic hyphomycete. Conidiophores hya-line, acroauxic, macronematous, mononematous,caespitose or solitary, initially straight but latergeniculate, unbranched. Conidiogenous cells tretic,integrated, terminal or becoming intercalary,cylindrical, proliferating several times sympodiallyor sometimes determinate. Tretoconidia solitary,aerogenous, hyaline, staurosporous, arms septate,radiating simultaneously from a swollen main body.

Porocladium aquaticum Descals sp.nov. (Figs.2D-G,4)

Cultura pura in 2% MA sub lumine diffuso ad 15°.Colonia 9 em uno mense crescens. Colonia compactacirca 4 mm profunda, atroschistacea, reversa vel eodemcolore vel atriore. Hyphae immersae 7-8!tm diam.Parietes pigmentiferi, densi. Mycelium aerium usquead 3 mm altum, totam coloniam tegens, laneum,murinum cum pannis albis. Sclerotia abundantia,atroschistacea vel atriora, mycelio aerio suffulta velimmersa, saepe confluentia, sphaerica vel lobata velmediae formae, circa 3 mm diam. Contenta pseudo-parenchymatosa, hyalina, ditia cellulis penariis.Cellularum fasciculatarum catenae moniliformes cumfrequentibus cellulis intercalaribus vel terminalibus,chlamydosporis similibus 17-35!tm latis. Sporulatiotypice aquatica. Conidiophore in aquam protrusa,hyalina, parietibus laevibus, macronematosa, mono-nernatosa, usque ad 500!tm longa, 3-4!tm lata;caespitosacum e sclerotiisexoriuntur velaliter dispersa,

primum recta, mox geniculata, sine ramis. Cellulaeconidiogenae hyalinae, treticae, in conidiophorisincorporatae, terminales sunt vel intercalares fiunt,cylindricae, circa 45!tm longae, paulum in apiceclavatae, Proliferatio sympodialis, cum 2-6 typicasuccessivisextensionibus. Conidia solitaria et acrogena,sed tandem extensionibus subapicalibus et sympodiali-bus detrusa. Conidia staurospora, hyalina, laevia cumcorpore principali conico truncato 30 x 16!tm adbasem, cum septo mediano transverso ante ramifica-tionern orto; protoplasma subtiliter granulatum.Brachia cylindrica, hyalina, 0-2 septata, 33-45!tmlonga, cum basi expansa, 6-8!tm lata, cum apicepaulum c1avato; simultaneo auctu crescentia; unumbrachium semper distale est, tria cetera paulum retroradiantia a cellula basali corporis principalis. Conidiaper rotundationem liberantur. Germinatio in agarotypice apicalis et vigorosa. Status perfectus ignotus.

Typi cultura deposita apud Herb IMI 199900 etHerb. Univ. Exeter no. 3206. Collectio: conidiumsingulum tetraradiatum a spuma in flumine Barle,Devon. boreal. Angliae.

Pure culture: (On 2 % malt extract agar at 15°under diffuse light.) Colony growth 9 em in onemonth. Colony compact, about 4 mm deep, dark-grey, reverse dark-grey to black. Immersed hyphae7-8,um diam. Walls warty, pigmented and thick.Aerial mycelium up to 3 mm high, covering theentire colony, woolly, mouse-grey with whitepatches. Sclerotia abundant, dark-grey to black,supported by the aerial mycelium or immersed;frequently confluent; spherical to lobed, about3 mm diam. Contents pseudoparenchymatous,hyaline, rich in storage globules. Monilioid chainsof cells in clusters, with frequent intercalary orterminal ovoid chlamydospore-like cells 17-35 pmbroad. Sporulation typically aquatic. Conidio-phores protruding into the water, hyaline, smooth-walled, macronematous, mononematous, up to500 pm long x 3-4 pm wide, caespitose whenarising from sclerotia or otherwise scattered,initially straight but later geniculate, unbranched.Conidiogenous cells hyaline, tretic, integrated,terminal or becoming intercalary, cylindrical,about 45 pm long, slightly clavate at the apex.Sympodial proliferation, with typically 2-6successive extensions. Conidia solitary and aero-genous but eventually pushed aside by the sub-apical sympodial extensions. Conidia stauros-porous, hyaline, smooth, with a truncate-conicalmain body 30 x 16 um at the base, with a mediantransverse septum developing prior to branching;protoplasm finely granular. Arms cylindrical,hyaline, 0-2 septate, 35-45 pm long, broadlybased, 6-8 pm wide, with the apex slightlyclavate; developing simultaneously; one armalways distal and the other three radiating slightlybackwards from the basal cell of the main body.


100JlIll

17:00(20'6)

H

15:00(16'6)

A

Fig. 4. Porocladium aquaticum from pure culture. A-H, Ontogenetic sequence (numbers indicate sequenceof septation); I, Mature, detached conidium.

E. Descals and others 21 3Conidia released by rounding-off. Germination onagar typically apical and vigorous .

This fungus does not appear to have been named,although Iqbal (1972) briefly described and illu-strated a similar conidium from foam in the R.Creedy in South Devon.

Its natural substrate and hab itat remain un-known, but normal sporulation in culture occursonly when submerged in a constant flow of clean,sterile, distilled water. In aerated but unchangedsterile distilled water, sporulation is scanty andbrief, with abundant misshapen conidia .

On LCA, a synthetic medium used by Miura &Kudo (1970), the fungus produces a few tretic,thick-walled slightly pigmented conidia withconspicuous attachment scars. They appearscattered over the colony and generally arise fromthe aerial mycelium. Conidia can be straight, with3-4 transverse septa, 55-100 Jim long x 7-15 flmwide, strongly resembling Drechslera. In somecases, though, either the apical or one or twolateral arms may also be present. Germination isfrom any cell, giving rise to either germ tubes orconidiophores with tretic conidiogenous cells. Aninteresting feature is the presence of abundantclusters of plate-like, brown crystals about 20 Jimlong on the surface of the hyphae.

Imperfect states with tretic conidiogenous cellsproliferating sympodially are unknown amongdescribed aquatic or waterborne hyphomycetes,but they are quite common among dematiaceousterrestrial forms (Ellis, 1971). Although wallpigmentation of conidia and supporting structuresis still considered a fundamental taxonomiccharacter in Fungi Imperfeeti there are strikingdevelopmental similarities between some of themand our genus. Alternaria, for example, includesspecies with solitary conidia on tretic conidio-genous cells (Ellis, 1971). P. aquaticum does not fitin it exclusively on grounds of conidium mor-phology and pigmentation. Several other demati-aceous genera with acropleurogenous tretoconidiaare otherwise very similar to Porocladium but thisspecies has not been noticed to produce in culturemore than one conidium at anyone pore. Acro-conidiella Lindquist & Alippi (Ellis, 1971) closelyresembles it too, except for its parasitic nature andunbranched conidia.

On fully developed conidiophores, conidio-genous cells appear to be monotretic, but it isinteresting to note that they consistently proliferateand give rise to the subsequent tretoconidium wellbefore a septum is laid down between the twoconidia (see sequence of septation in Fig. 4)·

Prol iferation is typically sympodial underconstant-flow conditions. Percurrent growths atvarying levels of the conidiophores may also be

seen, especially when sporulation takes place inunchanged water . But sympodial extensions arethen also present. These are believed to be twounrelated processes, percurrent growth takingplace from any cell on the conidiophore after anarrest in development, while sympodial extensionis a genetically fixed means of proliferation affect-ing conidiogenous cells.

BRACHIOSPHAERA Nawawi gen.nov.

Fungi Imperfecti, Hyphomycetes. Mycelium ex hyphisfuscis, septatis, ramosis compositum. Conidiophorasimplicia vel raro ramosa, macronematosa vel semi-macronematosa, erecta vel flexuosa, septata. Cellulaeconidiogenae terrninales, holoblasticae, sympodiales velraro percurrentes. Conidia sphaerica, et 4-7 brachiisradiantibus, septatis, simultaneis praedita.

Species typica: Brachiosphaera tropicalis Nawawi.

Resembling Actinospora in conidial morphologybut differing fundamentally in the method ofconidial production.

Brachiosphaera tropicalis Nawawi sp.nov.(Figs . 5, 6)

Coloniae in 2 % MA, rufobrunneae, cum mycelio raroaerio. Hyphae pallidae vel coloratiores usque adcolorem fuscum, plerumque grcssae, usquae at 12 flmdiam, saepecatenasmoniliformes cellularumformantesparietibus densis munitarum et globulis numerosisrefractivis sphaericis plenarum, Conidiophora simpliciavel raro rarnosa, primum erecta, mox f1exuosa fiunt;septata, ad basem colore pallide brunneo, supraincoloratafiunt; usque ad 500 pm longa, 9-12 pm lata.Cellulae conidiogenae terrninales, ovoideae vel dolii-formes vel rnixtae, 17-30 flm longae, 11-16 pm latae,hyalinae, forma sympodiali vel aliquando percurrenti.Conidia sphaerica vel globosa vel mixtae et mediaeformae, 46-58 pm diam, plena globulis rotundisrefractivis et 4-7 brachiis praedita, 95-180 flm longis,9-11 flm in loco latissimo latis, in apice rotundato ad4-5 pm paulatim contraetis, 3-5 septatis et 3-5 flm inaffixionis constrietione.

A spumae specimine seiuncta Pulau Langkawi,Malaysia mense Augusto 1972. Typus IMI 195180 etHerb. Exeter No. 3207.

Conidia of this fungus were frequently encoun-tered in most foam samples collected in Malaysia.They germinated readily on 2 % MA producing amoderately fast growing colony of 57 mm diamafter 35 days at 25°, which is the optimum tem-perature for growth and sporulation. The vege-tative mycelium is mostly immersed, light brownat first, becoming dark brown to black with age.Aerial mycelium is scanty, greyish, reverse black.No coloration of the medium occurred. Hyphaeare septate, thick-walled, 6-12 flm wide, pig-mented to varying degrees with age, smooth to

2 14 Actinospora and related fungi

Fig. 5. (A-D). Brachiosphaera tropicalis. Portion of conidiophores showing spent conidiogenous cells (A).with cellarette-like scars. The new conidiogenous cell at the apex has proliferated sympodially from thelast. (B). A nearly mature conidium still attached to the conidiophore. Note the sympodial proliferationof the new conidiogenous cell. (C). A conidiophore with 3 conidia. (D). Typical conidia from culture.(E-K) Clavariana aquatica. (E-H). Successive stages in the development of a conidium over a 3 h period.(1). A typical conidium from culture. (J). Two 'pycnidia' formed on agar. (K). 'Pycnidiospores'.


Fig. 6. Brachiosphaera tropicalis. A-E, Stages in conidial formation arranged in a developmental series;F-H, conidiophores showing detachment scars and proliferation; I, Conidia from submerged culture.

minutely tuberculate, the cells often becomingmonilioid and filled with numerous spherical,refractive globules. Malt agar is a poor mediumfor sporulation, but the fungus sporulates readilyat 20-25 ° on GCMA. Ncar u.v.-light has no

marked effect on growth or sporulation. The fungussporulated more readily when thin slices of agarcultures cut out from the central regions of colonieswere immersed in water.

The conidiophores arise as erect hyphae,


Table 1. Comparison of morphology of conidia ofBrachiosphaera jamaicensis and B. tropicalis

obvious. The mature conidia of A. megalospora aresubglobose to obpyriform but not fully sphericalas in B. tropicalis. Due to this shape it is oftenpossible to distinguish the terminal arm in aconidium of A. megalospora and hence the approxi-mate position of its attachment to the conidio-phore. This is not obvious in conidia of B.tropicalis. Further the arms in A. megalospora arebroad-based while in B . tropicalis each arm isconstricted at its attachment point. In all prob-ability the fungus described as A . megalospora byGoos (1970) from Hawaii is a Brachiosphaera.Comparisons of their cultural characteristics,conidium ontogeny and morphology given by Goosindicates the similarity between the two isolates.The conidia illustrated by Ingold (1973, Fig. 1A)and Miura (1974, Fig. 3) may also belong to thepresent fungus.

Actinosporajamaicensis Crane & Dumont (1975)has been described from material on wet wood inthe Tropics. Type material kindly sent to us (bythe authors) clearly shows the holoblastic natureof the conidia, which arise on conidiophoresproliferating sympodially (see Fig. 7). The generalappearance of the fungus suggests that we aredealing with a species of Brachiosphaera. Sizerelationships, the widely differing number ofconidial arms (see Table 1) and cultural characterslead us to believe that the Jamaican fungus is quitedistinct from B. tropicalis. We therefore propose anew combination, Brachiosphaera jamaicensis(Crane & Dumont) Nawawi (syn. Actinosporajamaicensis Crane & Dumont, Can .]. Bot. S3: 843,1975)·

A comparison between B. tropicalis and B.jamaicensis in Table 1 shows their differences.

Recently one of us . (A.N.) collected severalsamples of foam from two localities in Malaysia,

B . jamaicemis

Central sphere 55-66 pm

indistinguishable at first from the vegetativehyphae. They are hyaline, unbranched, septateand of variable length. As they lengthen, the cellsat the distal portion become slightly constricted attheir septa. The end cell gradually becomes sub-globose and finally spherical. When it reaches 30-40 p,m diam, 4-7 arms (usually 4) appear simul-taneously as blown out buds, and elongate rapidly,becoming 3-5 septate (Fig. 6A-D). The top armalways lies along the axis of the conidiophore whilethe rest develop from around the circumference ofthe sphere equidistant from one another. Out of150 conidia counted at random 107 were with 4arms, 35 with 5 arms, 7 with 6 arms and 1 with 7arms.

As the conidium matures, another ovoidconidiogenous cell develops very close to theattachment point and very soon produces anotherconidium (Fig. 6D, E). This process of sympodialproliferation is repeated until up to 10 or moreconidia are produced (Fig. 6H). The conidia ifundisturbed often remain attached to theirrespective conidiogenous cells. Occasionally aconidiogenous cell may produce 2 conidia suc-cessively before proliferating. A small, circular toirregular, collarette-like scar is clearly visible aftereach conidium is released . Under submerged,turbulent conditions, the conidia become detachedreadily and the conidiogenous cell appears toproliferate percurrently, producing successiveconidiogenous cells and conidia. In such cases it isdifficult to distinguish between sympodially andpercurrently produced conidiogenous cells as thescars are sometimes irregular and wide (Fig. 6G).

The mature conidia of B. tropicalis (Fig. 61)consist of a spherical body, 46-58 p,m diam andfilled with numerous small spherical globules upto 6 p,m diam. The spherical body is yellowishbrown and furnished with 4-7 radiating arms, 95-180 p,m long x 5)-11 p,m at the widest point,tapering to 4-5 p,m at the rounded apex, constrict-ing to 3-5 p,m at the points of origin and becoming3-5 septate. The spherical body is multi-nucleateand each cell of the arms contains up to 8 nuclei.

Brachiosphaera differs from Actinospora inseveral respects. Whereas the conidiophores inBrachiosphaera are unbranched and proliferatesympodially or (rarely) percurrently, in A ctino-spora branching of the conidiophore is invariablydichotomous and quite characteristic. Develop-mentally, too, they are different. It is difficult todistinguish between conidia of the two fungicollected from foam samples and indeed manyreports of A . megalospora from tropical or sub-tropical regions may have been based on mis-identifications. When the conidia of the two fungiare examined together the differences are quite

ArmsLength

Shape

Number

31-40 p,m8--9'5p,mThe arms areslightly bulbousafter theconstriction,mostly r-septateat maturity,conspicuouslytapering

With up to10-13 arms

B . tropicalis

45-58 pm(generally smaller)

95-180 p,m!)-11p,m

Arms not bulbous,taperinggradually,3-5 septate,mostly 3

Mostly with4-5 arms


Fig. 7. Brachiosphaera jamaicensis. Drawn from isotype material deposited at the University of Exeter(Exeter 3209). A-D, Conidiophores showing sympodial proliferation and the blastic nature of conidia;E-G, Detached conidia showing range in number of arms.

hoping to find conidia resembling A. jamaicensis.No conidia were found with 10-13 arms which areshort and obpyriform. Most conidia encounteredwere with 4-5 long, septate arms, typical of thosewhich were produced in culture of B. tropicalis.

Clavariana Nawawi gen.nov.

Fungi Imperfecti , Hyphomycetes. Mycelium ex hyphisfuscis septatis ramosis compositurn, quae conidiophoravelut ramos laterales ferunt. Conidiophora simplicia velin summo ramosa, septata, semimacronematosa.Cellulae


5 p.m. 6 p.m. 7 p.m. 8 p.m.

Fig . 8. Clavariana aquatica. A-H, A conidiophore drawn at intervals over a period of 8 h showing theformation of three complete conidia in a continuous flow chamber ; I, Range of conidiophore structure;J, Typical conidia from submerged culture.

conidiogenae monoblasticae vel polyblasticae, sympo-diales. Conidia sine colore, terminalia, obovoidea, tribustenuibus brachiis coronata cum brachia quarto basaliper separationis cicatricem statim post conidii separa-tionem forrnato.

Species typica: Clauariana aquatica sp.nov,

Clavariana aquatica Nawawi sp .nov. (Figs. 5, 8)

Coloniae in 2 % MA, rufobrunneae, velutinae cummycelio raro aerio . Hyphae pallidae vel fusciores usque

ad rufobrunneum colorem, grossae, usque ad 10 pmlatae . Conidiophore exorientia velut rami lateraleshypharum mycelialium, simplicia vel in summaramosa , hyalina, 80-400 pm longa, 5-9 pm lata.Cellulae conidiogenae breves, cylindricae vel clavatae velformae rnixtae et mediae, 29-35 pm longae, 7-10 pmlatae , monoblasticae et polyblasticae, sympodiales.Conidia ambitu obovoidea vel triangularia vel med ia etrnixta, basi 5-8 pm lata, supra autern latiora usque ad15-22 pm fiunt, 24-33 pm alta, coronata tribus brachiissimultaneis, 53-160 pm longis, 3-5 pm latis, 0-2


septatis, in apice ad 2-2'5 pm paulatim contractis.Brachium quarturn eiusdem mensurae per separationiscicatricern fit post conidii liberationem.

A spumae specimine segregata ad flumen UluGombak, Malaysia, mense Maio 1973. Typus 1M1195181 et Herb. Exeter No. 3208.

This fungus originates from a single conidiumisolated on 2 % MA from a foam sample collectedalong the Ulu Gombak river at the University ofMalaya Field Studies Centre. The conidia are ofrare occurrence and have never been observedfrom other foam samples. Conidia germ inatereadily on agar producing a germ tube from theextremity of each arm. A 35-day old colony on MAat 250 averages 65 mm diam, its optimum tem-perature being around 220. The colony is reddish-brown to brown, velvety with the vegetativemycelium mostly immersed, aerial myceliumscanty and the medium becoming discolouredwith a reddish tinge. The hyphae are thick-walled,pale to reddish-brown, septate and up to 10 pmwide. On GCMA at 25° numerous black, pycnidia-like bodies (Fig. 5]) measuring 75-120 pm long x52-112 pm wide, producing abundant minutespores are formed in concentric zones around theinoculum. These spores (3- 4 pm x 2-3 pm) werenever observed to germinate and arc presumablyspermatial in nature (Fig. 5 K). No sporulationwas observed on any of the media tested even whensubmerged in water . Later it was found that onlyolder portions of colonies on GCMA especiallyfrom around the inoculum, sporulate readily whensubmerged and incubated at 15-200.

Conidiophores arise as lateral branches of thevegetat ive hyphae. They are hyaline, septate,unbranched, producing a terminal conidium, ormore commonly consisting of an unbranched part80-400 pm long, 5-9 Ilm wide, which, towards itsfree end branches to produce a group of 2-3phi alide-like conidiogenous cells, each measuring29-35 pm long, 7-10 pm wide (Fig. 8 I). Each mayproduce up to three conidia successively fromdifferent areas of the cell before another conidio-genous cell similar in shape and size proliferatessympodially. Sometimes proliferation takes placethrough the detachment scar. Each conidiophoremay proliferate and produce up to 11 conidia .

The clavate conidial primordium is cut off earlyfrom the conidiogenous cell by a septum. Itenlarges rapidly in size becoming more or lesstriangular in outline. When the main body of theconidium is fully grown, three narrow divergingarms develop from its crown, growing outsimultaneously. When the arms reach 10-15 pmlong, the conidium is released by a break in thewall between conidium and conidiogenous cell,leaving a circular , cellarette-like scar on the apex

of the conidiogenous cell, but no such portion isvisible on the conidium. Immediately after releasethe fourth arm of the conidium develops throughthe detachment scar and very quickly attains thesame length as the rest of the arms. A septumdevelops at the base of each arm. The majority ofthe arm s are aseptate, but some may become 1-2septate. While the development of this conidiumis proceeding, another conidial primordium isbeing formed and grows to maturity in the sameway. The successive development of three conidiais shown in Fig. 8A-H.

The main body of the conidium (Fig. 8J) is5-8 Ilm wide at the base, broadening to 24:"33 /lmabove. The arms are more or less of the same lengthmeasuring 53-160 /lm long, 3-5/lm at its widestpoint and tapering to 2-2'5 /lm at the apex. Withage the central body becomes highly vacuolated.

The present fungus, which combines suchfeatures as monoblastic and polyblastic conidio-genous cells, and holoblastic conidia with fourradiating arms, the last basal arm appearing at alater stage of development, cannot be satisfactorilyplaced in any of the described genera of aquatichyphomycetes. It seems to be closely related toBrachiosphaera described above, especially in itscultural characteristics and conidial morphology,but the similarity ends there. Developmentallythey are quite distinct. A superficially similar genusis Clauariopsis De Wildeman which producesbroadly clavate conidia crowned by three long,narrow, radiating arms, but without the basal arm.

Conidia apparently belonging to this fungushave been recorded before by Ingold (1959, Fig.1G) from Nigeria and more recently by Miura(1974, Fig. 17). The Japanese spore is depictedwith the basal arm slightly displaced to one side.

Although the exact habitat and substrates ofthe three new fungi are at present unknown, thereis no doubt from the evidence available that theyare aquatic or growing in close vicinity of water.Based on conidial morphology alone it is quitetempting to suggest that the four fungi describedabove are congeneric. Basically their conidiaconsist of a central body, obpyriform, broadlyclavate to spherical in shape, bearing usually 4 long,radiating arms. Even some aspects of their develop-ment, too, show similarity. Each conidium beginswith the development of the central body, whichis its most obvious structure, followed by theformation of its arms, which arise simultaneously,except in Clavariana where the basal arm developsat a later stage. In other details of their conidio-phore apparatus and conidial ontogeny they aresufficiently distinct to warrant their placement indifferent genera. The only confusion that may


Actinosporamegalospora

Brachiosphaera tropicalis

Claoariana aquatica

Fig. 9. Diagram of types of conidiophores and conidia in the four genera described indicating how theyare different. The numbering of the conidia indicates their developmental sequences (not to scale).

E. Descals and others

Table 2. Main conidial characters and sizes of the five fungi

221

Actinospora megalospora(After Ingold, 1952)

Brachiosphaera tropicalis

Brachiosphaera jamaicensis

Porocladium aquaticum

Clavariana aquatica

Central body

35-4 0 /lmobpyriforrn to subglobose

46-58 !tmglobose to spherical

(51'5)-55'5-66 /lm*globose to spherical

16 x 30 /lmobpyriform to broadly clavate, a-celled

15-22 ust: wide x 24-33 /lm highobpyriform to broadly clavate

Lateral arms

100-180 p.m x 8 /lm, 4- 8 arms , usually 4,0-3 septate

95-180 !tm x 9-11 tun, 4-7 arms, usually4, 3-5 septate

(13)-30-44 x 6'5-9-(10) /lm* , (7)- 10-14arms, (0)- 1-2- (3)-septate

35-45 Itm x 6-8 Itm, mainly 4 arms , 0-2septate

53-160 /lm x 3-5 psn, mainly 4 arms, 0-2septate

* Data from the type description have been rounded off to the nearest half /lm.

arise is between conidia of Actinospora andBrachiosphaera when these are to be identifiedfrom isolated conidia found in foam samples.Contrary to the belief of Nilsson (1964) and Jones(1974) it is suspected that Actinospora is temperatein distribution while Brachiosphaera is morecommon in the tropical regions. Table 2 shows themain conidial characters and sizes of the five typespecies described above. F ig. 9 illustrates theirbasic developmental features.

In conclusion it must be emphasized thatassumptions on geographical distribution, eco-logical characteristics or simply floristic lists of atleast some of the lesser known waterborne hypho-mycetes should also be based on descriptionsderived from ontogenetic observations rather thanexclusively on morphology of detached conidiafound and preserved in foam and scum samples.

We are grateful to Professor C . T . Ingold andDr M. B. Ellis for their helpful comments. One ofus (AN.) is indebted to the Royal Society forfinancial assistance. We thank Professor F . Claytonfor providing the Latin diagnoses and Mr R. ADavey and Mr A G. Haley for technical assist-ance. Thanks are also due to Dr J. L. Crane(Illinois Natural History Survey, Urbana, Illinois)for providing type material.

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8

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(Accepted for publication 8 December 1975)

Documents

Developmental studies in Actinospora and three similar aquatic hyphomycetes