Costa Rican species of Nematoctonus(anamorphic Pleurotaceae)

Alexandra T.E. Koziak, Felipe Chavarria Diaz, Joel Diaz, Maria Garcia,Daniel H. Janzen, and R. Greg Thorn

Abstract: Nematoctonus (Hyphomycetes) and Hohenbuehelia (Agaricales, Pleurotaceae) are the names for the asexual andsexual stages of a genus of nematode-destroying fungi (Basidiomycota). Six morphospecies of Nematoctonus, all previouslydescribed, were isolated from fruiting bodies of Hohenbuehelia and from 439 samples of soil and organic debris collected inall 12 Holdridge life zones in Costa Rica. Nematoctonus was recorded in all but three life zones at the lowest and highest alti-tudes: tropical dry forest, tropical moist forest, and subalpine rain paramo. Isolates of Nematoctonus were identified by the mi-cromorphology of their conidia and adhesive knobs, which are usually an hourglass-shaped secretory cell surrounded by adrop of mucilage. Adhesive knobs were found either exclusively on hyphae in predatory species (Nematoctonus robustus F.R.Jones), exclusively on germinated conidia in parasitoid species (Nematoctonus leptosporus Drechsler, Nematoctonus pachy-sporus Drechsler, and Nematoctonus tylosporus Drechsler) or on both hyphae and germinated conidia in a group we term ‘‘in-termediate predators’’ (Nematoctonus angustatus Thorn & G.L. Barron, Nematoctonus geogenius Thorn & G.L. Barron, andone monokaryotic isolate of N. robustus). Teleomorph–anamorph connections, made by culturing the anamorph from a teleo-morph fruiting body, were made for N. angustatus (Hohenbuehelia angustata (Berk.) Singer), N. geogenius(Hohenbuehelia petalodes (Fr.) Schulz.), N. leptosporus (an unidentified Hohenbuehelia), and N. robustus(Hohenbuehelia grisea (Peck) Singer).

Key words: Nematophagous fungi, anamorph, teleomorph, biotic surveys and inventories.

Resume : Les noms de Nematoctonus (Hyphomycetes) et Hohenbuehelia (Agaricales, Pleurotaceae) designent respective-ment les stades asexuel et sexuel d’un genre de champignons (Basidiomycota) destructeurs de nematodes. Les auteurs ontisole six especes morphologiques de Nematoctonus, toutes deja decrites auparavant, a partir de fructifications du Hohen-buehelia, et de 439 echantillons de sol et de debris organiques recoltes sur l’ensemble des 12 zones biologiques de Hol-ridge, au Costa Rica. Les auteurs ont repere le Nematoctonus dans toutes, sauf 3 de ces zones, aux altitudes les pluselevees et les plus bases : foret tropicale seche, foret tropicale humide et foret ombrophile subalpine de Panamo. La micro-morphologie des conidies et les boutons adhesifs ont servi a identifier les isolats de Nematoctonus; les cellules secretricesde ces boutons adhesifs ont generalement la forme d’un verre de montre et sont surmontees d’une goutte de mucilage. Onretrouve les boutons adhesifs exclusivement sur les hyphes des especes predatrices (Nematoctonus robustus F.R. Jones),uniquement sur les conidies germees chez les especes parasitoıdes (Nematoctonus leptosporus Drechsler, N. pachysporusDrechsler, et Nematoctonus tylosporus Drechsler), ou a la fois sur les hyphes et les conidies germees, chez un groupenomme par les auteurs « predateurs intermediaires » (Nematoctonus angustus Thorn & G.L. Barron, Nematoctonus geoge-nius Thorn & G.L. Barron, et un isolat monocaryote du N. robustus). Les relations teleomorphe-anamorphe, realisees parla mise en culture de l’anamorphe a partir de la fructification teleomorphe, ont ete determinees pour les N. angustus(Hohenbuehelia angustata (Berk.) Singer), N. geogenius (Hohenbuehelia petalodes (Fr.) Schulz.), N. leptosporus(un Hohenbuehelia indetermine) et le N. robustus (Hohenbuehelia griseus (Peck) Singer)).

Mots-cles : champignons nematophages, anamorphe, teleomorphe, survols et inventaires biologiques.

[Traduit par la Redaction]

Introduction

The genus Nematoctonus was described by Drechsler(1941) for two species of nematode-destroying fungi thatwere distinguished by the possession of clamp connections(hyphal loops bridging septa), which are characteristic ofphylum Basidiomycota. Drechsler (1941) compared his new

fungi to illustrations of the production of conidia (asexualspores) by the basidiomycetes Galzinia incrustans (Hohn. &Litsch.) Parmasto (Lyman 1907; Nobles 1937) and Hohen-buehelia sp. (Vandendries 1934, as Pleurotus pinsitus (Fr.)Sacc.). However, since neither basidia nor basidiospores ofa sexual form (teleomorph) were produced in his cultureson maize-meal agar with nematodes, Drechsler (1941) de-

Received 9 March 2007. Published on the NRC Research Press Web site at canjbot.nrc.ca on 17 September 2007.

A.T.E. Koziak and R.G. Thorn.1 Department of Biology, University of Western Ontario, London, ON, N6A 5B7, Canada.F.C. Diaz, J. Diaz, and M. Garcia. Area de Conservacion Guanacaste, Apartado Postal 169-5000, Liberia, Guanacaste, Costa Rica.D.H. Janzen. Department of Biology, University of Philadelphia, Philadelphia, PA 19104, USA.

1Corresponding author (e-mail: rgthorn@uwo.ca).

749

Can. J. Bot. 85: 749–761 (2007) doi:10.1139/B07-049 # 2007 NRC Canada

scribed Nematoctonus as an asexual (anamorph) form-genusof the deuteromycetes.

Nematoctonus is one of the few genera of nematophagousfungi in which individual species may attack via mycelialtraps or separate adhesive spores (Barron 1977), and thisdistinction is useful in their identification. Since multiple in-dividual animals are killed and consumed as food (not dis-eased), the nematophagous fungi are predators, notpathogens; those that attack via adhesive spores may betermed parasitoids. Parasitoids, whether insects or fungi, arepredators that delegate the killing of the prey to their off-spring or propagules. For instance, the larva of a parasitoidwasp consumes the caterpillar in which its egg was laid byits mother, and the caterpillar usually dies either as the larvaexits to pupate, or soon afterwards (Godfray 1994). In nem-atophagous fungi and the ecologically similar entomophilousfungi, conidia produced by the parent mycelium infect suc-ceeding generations of prey. In Nematoctonus, once a coni-dium has attached, a penetration peg enters the nematodeand assimilative hyphae colonize, digest, and absorb thebody contents. Fertile hyphae emerge to produce more con-idia on a mycelium of limited extent. In evolutionary terms,each ramet of a parasitoid Nematooctonus is restricted toone nematode, but the genet or evolutionary individual(Janzen 1977) is extensive, dispersed, and consumes manynematodes.

A ramet of a predatory Nematoctonus species produces anextensive mycelium and captures many nematodes withhourglass-shaped adhesive knobs on the hyphae. Nematodesbecome attached to these adhesive knobs and the cuticle ofthe nematode is penetrated by the infective hyphae. Once in-side the host, assimilative hyphae form to digest and absorbthe contents of the nematode. The absorbed nutrients areused to produce additional fertile hyphae with adhesiveknobs and conidia (Drechsler 1946; Thorn and Barron1986). As with parasitoid species, the genet or evolutionaryindividual may consist of multiple, dispersed ramets.

Barron and Dierkes (1977) were the first to document theproduction of a fruiting body of the genus Hohenbuehelia byan unidentified predatory species of Nematoctonus, whichwas later identified as the Hohenbuehelia grisea (Peck)Singer teleomorph of Nematoctonus robustus F.R. Jones(Thorn and Barron 1986). Thorn and Barron (1986) wereable to connect 13 Hohenbuehelia species to their Nematocto-nus anamorphs by culturing teleomorphs to produce ana-morphs in culture. Most of these species were predatory, withthe exception of two parasitoids, the Nematoctonus cylindro-sporus Thorn & Barron anamorph of Hohenbuehelia auris-calpium (Maire) Singer and the Nematoctonus pachysporusDrechsler anamorph of Hohenbuehelia izonetae Singer(1989). Several related Hohenbuehelia species had the sameNematoctonus anamorph form-species: the anamorphs ofHohenbuehelia approximans (Peck) Singer, Hohenbueheliaatrocaerulea (Fr.) Singer), Hohenbuehelia cyphelliformis(Berk.) O.K. Mill., H. grisea, and Hohenbuehelia nigra(Schwein.) Singer were all identified as N. robustus; theanamorphs of Hohenbuehelia pinacearum Thorn andHohenbuehelia unguicularis (Fr.) O.K. Mill. were identi-fied as N. brevisporus Thorn & G.L. Barron; and the ana-morphs of Hohenbuehelia petalodes (Fr.) Schulz. andHohenbuehelia tremula (Schaeff.) Thorn & Barron were

identified as Nematoctonus geogenius Thorn & G.L. Bar-ron (Thorn and Barron 1986). It was suggested that thiswas due to the larger number of morphological character-istics available for recognition of species in Hohenbueheliacompared to Nematoctonus (Thorn and Barron 1986); thatis, form-species in Nematoctonus are more inclusive.

Species of Nematoctonus have been found throughoutnorthern temperate areas of the world, including the UnitedStates, England, Finland, Canada, Ireland, Sweden, Japan,and Germany (Drechsler 1941, 1943, 1946, 1949, 1954;Duddington 1951; Ruokola and Salonen 1967; Salonen andRuokola 1968; Giuma and Cooke 1971; Barron 1978; Grayand Duff 1982; Gray 1984; Saikawa and Arai 1986; Thornand Barron 1986; Durschner-Pelz 1987; Bailey and Gray1989; Stadler et al. 1994). Nematoctonus has also been re-ported from Queensland in Australia (McCulloch 1977, trop-ical to subtropical), New Zealand (Fowler 1970, temperate),and Taiwan (Liou and Tzean 1997, subtropical), and twospecies have been described from Africa: N. robustus fromGhana (Jones 1964, tropical) and Nematoctonus tripolitaniusGiuma and R.C. Cooke, from Libya (warm temperate).Nematoctonus has not been studied in the neotropical bio-geographic region. There are a few published reports ofnematode-destroying fungi from neotropical soils or organicdebris (Gazzano 1973, from Uruguay; Gamundi and Spinedi1982, from Argentina; Bucaro 1983, from El Salvador;Lappe and Ulloa 1983, from Mexico; Morgan-Jones et al.1984, from Colombia; Rubner 1994, from Ecuador; Pers-mark et al. 1995, from Panama, Costa Rica and Nicaragua;and Dalla Pria et al. 1991, Naves and Campos 1991; Santoset al. 1991; Dias et al. 1995; and Saumell et al. 2000, allfrom Brazil). Among these, the only records of Nematocto-nus are of N. robustus from Minas Gerais, Brazil (22.58S,natural vegetation a subtropical moist forest, Saumell et al.2000) and N. leiosporus from Punta Carretas, near Montevi-deo, Uruguay (34.98S, in a warm temperate grassland orpampas, Gazzano 1973). There has only been one previousreport of nematode-destroying fungi from Costa Rica basedon just two Costa Rican soil samples from which only fivespecies were reported, with no records of Nematoctonus(Persmark et al. 1995).

Costa Rica is a country of 51 060 km2 between Nicaraguaand Panama (*98–118N latitude) and has tremendous diver-sity in vascular plants, vertebrates, and the selected groups ofinvertebrates that have been studied (Janzen 1983). The richlyvaried topography in Costa Rica produces 12 Holdridge LifeZones (Holdridge 1971); a system of ecosystem categoriza-tion based on precipitation, altitude, and temperature), corre-lated with the great biodiversity in this small country.Costa Rica is much richer in species than temperate regionsof the world and is estimated to harbour over 500 000 spe-cies. Of these, 84% still are unknown to science. It hasbeen estimated that only 2% of Costa Rica’s fungi aredocumented. The Costa Rican Instituto Nacional de Biodi-versidad (INBio) was established in 1989, to create a na-tional effort for conservation, knowledge, and sustainable useof biodiversity (www.inbio.eas.ualberta.ca/en/default2.html).The Costa Rican national biodiversity inventory is focused onarthropods, plants, mollusks, and fungi, with inventory effortconcentrated in five conservation areas (A.C.): A.C. Arenal-Tilaran, A.C. La Amistad Pacifico, A.C. La Amistad Caribe,

750 Can. J. Bot. Vol. 85, 2007

# 2007 NRC Canada

A.C. Tempisque, and A.C. Osa (www.inbio.ac.cr/papers/gt_Hongos/es/index.htm) and, prior to 1999, A.C. Guanacaste.

The objective of this study was to discover and identify asmany species of Nematoctonus as possible from Costa Rica.We did this by sampling organic debris and soil from the 12Holdridge Life Zones in Costa Rica to isolate Nematoctonusvia bait nematodes, and by searching for fruiting bodies ofHohenbuehelia species (teleomorphs) to culture their Nema-toctonus anamorphs. Only one species of Hohenbuehelia,H. nigra, has been reported from Costa Rica (Ovrebo1997), and no details of its anamorph have been reported.Several members of the H. nigra species complex havebeen reported from neighbouring Panama, from Cuba, andfrom Argentina (Alberto et al. 1998; Fazio and Alberto2001). Several other, unidentified species of Hohenbueheliaare in the INBio herbarium (R.G. Thorn, unpublished data,2001). Lack of a monograph of the neotropical species ofHohenbuehelia made it impractical to include the identifica-tion of Hohenbuehelia teleomorphs at this time.

Materials and methods

Sampling for Nematoctonus and HohenbueheliaA total of 439 samples (approximately 150–250 mL) of

soil, rotting wood, moss and other organic debris were col-lected during 2001–2004, in each of the 12 Holdridge lifezones in Costa Rica (Table 1). Sampling trips were of oneto three weeks duration, mostly during the wet months fromJune to November, and few localities were sampled in morethan one season. Samples were collected in sterile polyethy-lene bags and stored at 5 8C until processing. While collect-ing these samples, an attempt was made to locate and collectas many specimens as possible of Hohenbuehelia, fromwhich cultures of Nematoctonus were derived.

Isolation techniques for NematoctonusThe soil-sprinkling technique (Barron 1977, see below)

was chosen as the most effective technique to isolate Nema-toctonus from soil or organic debris because of the verysmall numbers of nematodes encountered in most samples(R.G. Thorn, unpublished data, 2001). This made the obser-vation of nematodes recovered from samples using the Baer-mann funnel technique (Barron 1977) unproductive, sincefar too few nematodes were recovered to detect the smallproportion that might have been infected by Nematoctonus.Stock cultures of the bait nematodes Caenorhabditis elegans(Maupas) (order Rhabditida, suborder Rhabditina) and Pan-agrellus redivivus (L.), (order Rhabditida, suborder Cephalo-bina) together with their food bacteria, were maintained atroom temperature on 1.5% water agar (WA) plates. Nema-tode stock cultures were transferred weekly by washing astock plate with 2 mL of 0.85% saline solution and pipetting200 mL of nematode suspension onto each of 3–5 new stockplates of WA. Soil-sprinkle plates were prepared by mixingthe sample within the sampling bag, then dispersing approx-imately 0.5 g across the surface of WA plates that had beenbaited with 100–120 mL of nematode suspension two daysbefore sprinkling samples (Barron 1977; Thorn and Barron1986). Twenty samples were processed at a time and eachsample was sprinkled on 3 plates. All samples were proc-

essed at least twice, thus six or more plates per samplewere observed. Sprinkled plates were inspected twice aweek for eight weeks using a dissecting microscope at 10–45�. Nematoctonus species were confirmed under the com-pound microscope (100�, 200�, or 400�) by the presenceof clamp connections and hourglass-shaped secretory cellson their hyphae or conidia.

When nematodes infected by Nematoctonus were de-tected, the freshly infected nematodes were lifted using asterile glass transfer needle, then washed by gently draggingthem through three puddles of 0.85% saline (and the agarbeneath) on a clean WA plate (Thorn and Barron 1986).The needle was flamed and cooled with 95% ethanol be-tween each washing. The cleaned nematode was transferredto a plate of potato dextrose agar (PDA) containing 50 mg/Lchloramphenicol (Ant PDA). After hyphae emerged fromthe nematode, a pure culture of Nematoctonus was obtainedby transferring mycelium from the colony margin onto PDAwithout antibiotics. Isolates and unculturable records fromsoil samples are referred to by sample number (e.g., 03-RGTSN-519, found in sample number 519 from 2003).

Isolation and culturing of HohenbueheliaDikaryotic polyspore cultures were obtained from fresh

collections of Hohenbuehelia by suspending a small portionof Hohenbuehelia pileus to the lid of a 35 mm Petri dish ofmalt extract agar (ME) containing 50 mg/L chloramphenicoland 5 mg/L benomyl (MEB). Dikaryotic tissue cultures wereobtained by aseptically transferring a small portion of thefruit body tissue to a 35 mm Petri dish containing Ant PDAor MEB. Once a clean culture was established, a piece ofthe mycelium from the colony margin was transferred toPDA and ME plates (lacking antibiotics). Isolates from fruit-ing bodies are referred to by the collection number (e.g.,RGT 010810/02, the second collection on 10 August 2001).All cultures are preserved at UWO as slants on PDA andMEA under mineral oil.

Microscopy, measurements, and documentationMorphological features were studied on water agar with

nematodes (Thorn and Barron 1986). Cultures of Nematoc-tonus were grown to *3 cm on WA, when several drops ofbait nematodes were added. Plates were observed for fungal–nematode interactions after 24 h and for up to severalweeks. Infected nematodes and conidia, either from cul-tures or directly from the sprinkle plates, were transferredon small chips of agar to microscope slides and mountedin cotton blue (0.5% w/v in 85% lactic acid, Kirk et al.2001), and a drop of polyvinyl alcohol (Omar et al. 1979)was added for semi-permanent mounts.

Hyphae, conidia, and predatory adhesive knobs weremeasured and photographed at 1000�. For each isolate, 30ungerminated conidia were measured for length, width, andQ (ratio of length to width). When available, 10 germinatedconidia, germ tubes, hourglass cells, predatory adhesiveknobs, mucoid drops, aleuriospores, conidial pegs, assimila-tive hyphae, and fertile hyphae were measured for length,width, and Q. Measurements are reported as the 11th–90thpercentile, with the extremes in parentheses. Photographswere taken with a Nikon CoolPix 990 digital camera, andimages brought into Photoshop1 6.0 (Mac) or CS12 (PC)

Koziak et al. 751

# 2007 NRC Canada

(Adobe Systems Inc., San Jose, Calif.) for adjustment ofbrightness and contrast, and assembly of the plate. All de-scriptions are based on Costa Rican isolates examined duringthis study. General collection localities are listed in Table 1,with full details available online as Supplementary data.2

Results

Sprinkle plates of 439 samples of soil, rotting wood, moss,and other organic debris yielded 45 records of Nematoctonus,representing six previously described form-species (Table 1),plus two unidentified parasitoid species that could not be cul-tured. Nematoctonus pachysporus (18 records including 17isolates plus one that did not grow in culture), Nematoctonus-tylosporus Drechsler (1 isolate and 11 records that did notgrow in culture), and N. robustus (9 isolates) were mostcommon. The other species recorded from soil samples wereN. geogenius (1 isolate) and Nematoctonus leptosporusDrechsler (3 records). Four Nematoctonus species were recov-

ered from Hohenbuehelia fruiting bodies: N. robustus (8 iso-lates from H. grisea), Nematoctonus angustatus Thorn & G.L.Barron (4 isolates from Hohenbuehelia angustata (Berk.)Singer), N. geogenius (1 isolate from H. petalodes), and N. lep-tosporus (1 isolate from an unidentified Hohenbuehelia).

NematoctonusSixteen described species are characterized primarily on

the variation found in their conidia and adhesive processes.Assimilative hyphae are produced within the nematode; theseare hyaline, clamped, and 0.8–2.4(–3.2) mm in diameter. Fer-tile hyphae, produced outside the nematode, are similar andproduce holoblastic conidia with smooth, thin walls fromsmall, tapering pegs. Conidia are one-celled (aseptate) in allspecies except N. lignicola (nom. invalid., Salonen and Ruo-kola 1968), which is also not known to be predaceous. Con-idial pegs are simple, producing a single conidium at the tip,or are branched to produce two or three conidia. Characteristicadhesive processes with an hour-glass shaped secretory cell

Table 1. Sampling for and detection of Nematoctonus in Costa Rica. The name of the Life Zone refers to the undisturbed vegetation char-acteristic of a given altitudinal belt, temperature, and precipitation regime (Holdridge 1971).

Life zone, N n Example localities, coordinates Ha Hp Ng Hsp Nl Np Hg Nr Nt

Tropical dry forest, 14 4 Playa Naranjo* 10847’N 85841’W10 Hacienda La Pacıfica* 10828’N 85807’W

Tropical moist forest, 15 10 Puerto Jimenez 8832’N 83818’W3 Tilaran 10826’N 84854’W2 Volcan 9816’N 83833’W

Tropical wet forest, 121 24 La Selva 10826’N 84802’W 1 114 San Cristobal 10852’N 85824’W73 Sirena 8829’N 83836’W 1 510 Zapote 10829’N 83858’W

Premontane moist forest, 33 15 Los Almendros{ 11802’N 85831.7’W 25 Liberia-Sta Maria{ 10842’N 85829’W

13 Santa Rosa{ 10850’N 85837’W 1Premontane wet forest, 97 16 Las Alturas 8857’N 82850’W 2 4 1

7 Bijagua 10847’N 85805’W10 CATIE 9852’N 83839’W13 Rio Celeste 10841’N 85800’W15 Rio Chiquito 10840’N 85806’W18 Santa Maria 10846’N 85818’W 1 1 218 Sector Montezuma 10840’N 85803.6’W 1 1

Premontane rain forest, 65 33 Las Cruces 8847’N 82858’W 1 1 123 Heliconias 10842’N 85802’W 11 Cataratas La Paz 10812’N 84810’W8 Guayabo 9858’N 83841’W 1 1 1 1

Lower Montane moist forest, 5 5 Volcan Irazu *3000m 9858’N 83849.5’W 1 1Lower Montane wet forest, 38 11 Reserva Sta Elena 10819’N 84849’W 1 1 1

10 Volcan Barva *2600m 10807’N 84807.5’W 1 4 15 Volcan Poas *2000m 10809’N 84814’W 1 1

12 Volcan Turrialba *2500m 10800’N 83846’W 3 2Lower Montane rain forest, 22 22 San Gerardo de Dota 9833’N 83848’W 1Montane wet forest, 9 9 Volcan Irazu summit 9859’N 83851’W 1 2Montane rain forest, 10 10 Volcan Poas summit 10811’N 84814’W 1 3Subalpine rain paramo, 10 10 Cerro de la Muerte 9833’N 83846’W

Note: N and n refer to the number of samples collected in the Life Zone and locality, respectively. For province and conservation area of localities listed,see under ‘‘Isolates examined’’ in species descriptions. Ha, Hohenbuehelia angustata (Nematoctonus angustatus); Hg, H. grisea (N. robustus); Hp, H. petalodes(N. geogenius); Hsp, Hohenbuehelia sp. (N. leptosporus); Ng, N. geogenius; Nl, N. leptosporus; Np, N. pachysporus; Nr, N. robustus; Nt, N. tylosporus.*Moist province transition.{Basal belt transition.

2 Supplementary data for this article are available on the journal Web site (http://canjbot.nrc.ca) or may be purchased from the Depository ofUnpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ON K1A0R6, Canada. DUD 5167. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml.

752 Can. J. Bot. Vol. 85, 2007

# 2007 NRC Canada

surrounded by adhesive mucus are produced on fertile hy-phae, germinated conidia, or both. Three species produce twotypes of spores: thin walled spores (conidia) and thick-walledspores known as aleuriospores (resting spores, also known aschlamydospores) that are warty and elliptical or ovoid in

shape. Two species, N. leptosporus and N. tylosporus, are ap-parently specific to nematodes of the genus Bunonema (orderRhabditida, suborder Rhabditina). The other species of preda-tory or parasitic fungi attack various species of nematodes,although the natural prey of most species is unknown.

Key to the species of Nematoctonus. Species found in Costa Rica are indicated in bold and are described below; theremaining validly described species are included in the key since they may be discovered in Costa Rica1a Attacking nematodes only by means of adhesive conidia; no adhesive knobs present on fertile hyphae . . . . . . . . . . 21b Attacking nematodes by means of adhesive knobs on fertile hyphae (and sometimes also by adhesive germinated conidia)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2a (1a) Thickwalled, ornamented aleuriospores present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32b (1a) Aleuriospores absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3a (2a) Conidia fusiform, plump, 12–17 mm � 3–5 mm; on diverse nematodes . . . . . . . . . . . . . . . . . . . . N. pachysporus3b (2a) Conidia narrowly fusiform, 12–17 mm � 1.6–3.2 mm; only on Bunonema . . . . . . . . . . . . . . . . . . . N. tylosporus

4a (2b) Conidia narrowly fusiform, 17–24 mm � 1.6–2.4 mm; only on Bunonema . . . . . . . . . . . . . . . . . . N. leptosporus4b (2b) Conidia broader or more cylindric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5a (4b) Conidia fusoid and distinctly tapered, with a prominent hook at the tip, 12–24 mm � 2.7–5.4 mm . . . N. hamatus3

5b (4b) Conidia without a hooked apex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

6a (5b) Conidia fusoid, generally straight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76b (5b) Conidia more cylindric, or curved. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

7a (6a) Conidia 14–30 mm � 2.3–4.0 mm; hour-glass cell of adhesive knob with subapical constriction and nearly sphericalterminal swelling; attacking a broad range of nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. leiosporus

7b (6a) Conidia shorter, 11–18 mm � 1.6–3.2(–4.0) mm; only on Bunonema . . . . . . . . . . . . . . . . . . . . . N. tylosporus4

8a (6b) Conidia cylindric, 12–24 mm � 2.2–4.2 mm, straight to slightly curved . . . . . . . . . . . . . . . . . N. cylindrosporus8b (6b) Conidia more fusoid, occasionally elliptic-cylindric, and usually curved, (6.4–)9–16 mm � (1.6–)3.0–4.5 mm, germi-

nating to form one or two swellings prior to apical hour-glass cell. . . . . . . . . . . . . . . . . . . . . . . N. geogenius3

9a (1b) Aleuriospores present, 10–17.5 mm � 7.5–10 mm; conidia large and fusoid-elliptic, 15–25 mm � 5–7.5 mm . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. tripolitanius

9b (1b) Aleuriospores absent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

10a (9b) Adhesive knobs primarily terminal; hour-glass cells 3.5–6.5 mm long . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1110b (9b) Adhesive knobs primarily intercalary; hour-glass cells 6–14 mm long . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

11a (10a) Conidia plump, cylindric-elliptical, bean- or kidney-shaped, slightly or strongly curved, 9–14 mm � 3–5 mm . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. subreniformis

11b (10a) Conidia more slender, straight or strongly curved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

12a (11b) Conidia straight or a few slightly curved, 11–18 mm � 3.3–4.5 mm . . . . . . . . . . . . . . . . . . . . N. haptocladus12b (11b) Conidia mostly strongly curved, sausage shaped, 9–15 mm � 2.5–4.0 mm . . . . . . . . . . . . . . N. campylosporus

13a (10b) Majority of conidia strongly curved, sausage shaped, 7–15(–19) mm � 2.5–4.0 mm . . . . . . . . . . . . N. robustus13b (10b) Conidia straight or slightly curved, cylindric, elliptical or tapering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

14a (13b) Many conidia over 13 mm long . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1514b (13b) Most conidia under 13 mm long . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

15a (14a) Conidia cylindric-elliptical, 10–23 mm � 3.3–5.5 mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. concurrens15b (14a) Conidia fusoid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

16a (15b) Conidia with a prominent hook at the tip, 12–24 mm � 2.7–5.4 mm . . . . . . . . . . . . . . . . . . . . . . N. hamatus16b (15b) Conidia without an apical hook, 10–18 mm � 2 mm . . . . . . Nematoctonus anamorph of Hohenbuehelia longipes

17a (14b) Conidia cylindric and usually straight, 7–12 mm � 2–4 mm; hourglass cells of predatory adhesive knobs 2.4–3.2 mm broad . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. brevisporus

17b (14b) Conidia narrowly elliptic or fusoid and often curved near the tip, hourglass cells 2.4–5.6 mm broad . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3 Included here because predatory adhesive knobs may be overlooked.4 Isolates lacking aleuriospores.

Koziak et al. 753

# 2007 NRC Canada

18a (17b) Hourglass of predatory adhesive knob with pronounced waist; conidia cylindric or fusoid, straight or curved, 6.4–8.0 mm � 1.6–2.4 mm in Costa Rica isolates (but 7.5–15 mm � 3.0–4.5 mm in temperate isolates) . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. geogenius

18b (17b) Hourglass of predatory adhesive knobs without distinct waist; conidia cylindric or fusoid, straight or curved, 7.2–10.4 mm � 1.6–2.6(–3.2) mm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. angustatus

Extralimital speciesN. brevisporus (see 17a in the above key). This species

has only been isolated from its teleomorphs H. pinacearumand H. unguicularis, which are known from Europe andNorth America (Thorn and Barron 1986).

N. campylosporus Drechsler (see 12b in the above key).This species, known from Louisiana, USA (Drechsler1954), Queensland, Australia (McCulloch 1977), and Argen-tina (Thorn and Barron 1986) differs from N. robustus in itssmaller, terminal adhesive knobs. Hohenbuehelia portegna(Speg.) Singer has been reported as a teleomorph (Thornand Barron 1986).

N. concurrens Drechsler (see 15a in the above key). Thisspecies has been reported from Oregon (Drechsler 1949) andCalifornia (Jaffee et al. 1996) in the USA, and from theUK (Giuma and Cooke 1971).

N. cylindrosporus (teleomorphs H. auriscalpium and al-lies) (see 8a in the above key). This species has only beenisolated from fruiting bodies of its teleomorphs, which arefound on rotting deciduous and coniferous wood in Europeand North America (Thorn and Barron 1986).

N. hamatus Thorn & G.L. Barron (teleomorph H. mastru-cata (Fr.) Singer)(see 5a in the above key). This speciesknown from Europe, Japan, and North America (south tocoastal texas), may be expected in Costa Rica (Thorn andBarron 1986).

N. haptocladus Drechsler (see 12a in the above key). Thisspecies has been recorded from Colorado, USA (Drechsler1946), the UK (Giuma and Cooke 1971), and Nova Scotia,Canada (Mahoney and Strongman 1994).

N. leiosporus Drechsler (see 7a in the above key). Thisspecies is known from North and South America, Europe,and Australasia, and is common in farmyard soils. No tele-omorph is known (Thorn and Barron 1986).

Nematoctonus anamorph of H. longipes (Boud.)M.M. Moser (see 16b in the above key). This species isknown from boreal and alpine Europe and North America(Thorn and Barron 1986).

N. subreniformis Thorn & G.L. Barron (see 11a in theabove key). This species has only been recorded from well-rotted deciduous wood and soil in Ontario, Canada (Thornand Barron 1986).

N. tripolitanius (see 9a in the above key). This specieshas been recorded only from olive grove soils in Libya(Giuma and Cooke 1972).

Nematoctonus angustatus Thorn & G.L. Barron (1986)Figs. 1–2

Conidial pegs simple, most 1.6–4.0 mm � 0.8–1.6 mm.Predatory adhesive knobs predominantly intercalary; hour-glass cells 5–12 mm long � 2.4–5.6 mm wide at the base, not

prominently constricted; surrounded by an adhesive mucoiddrop 5–13 mm in diameter. Conidia narrow, cylindrical or fu-soid, straight or slightly curved, (5.6–)7.2–10.4(–12.8) mm �1.6–3.2(–4.0) mm, Q = (2.3–)3.0–4.5(–6.0); germinating toproduce a tapering germ tube with apical adhesive knobconsisting of an hourglass cell 2.4–4.0 mm long � 0.8–1.6 mm wide, surrounded by a mucoid drop 3–5 mm in di-ameter. Aleuriospores absent.ISOLATES EXAMINED: This species was cultured from four collec-tions of Hohenbeuehlia angustata found in premontane wetforest and lower montane wet forest (Table 1). Other collec-tions of H. angustata, not cultured, were found at Area deConservacion Guanacaste, Guanacaste Prov., Pitilla Biologi-cal Station, tropical wet forest premontane belt transition,10859’N 85826’W, ca. 700 m (RGT 960608/12 and 960610/02).SUBSTRATES: on soggy, brown-rotted logs and mossy stumps.

HABITAT: in primary forests with abundant deadfalls.

PREY: Panagrellus redivivus and unidentified Costa Ricannematodes.TELEOMORPH: Hohenbuehelia angustata (Berk.) Singer.

REMARKS: Nematoctonus angustatus is characterized by conidiathat are narrow, cylindrical to tapering, straight or slightlycurved and germinate to produce an hourglass cell sur-rounded by a mucoid drop and by predominantly intercalary,large predatory hourglass cells surrounded by a large adhe-sive mucoid drop. Predatory hourglass cells were observedto be mainly attached to the nematode’s head and subse-quent hyphae were observed to have approached and enteredthe dead nematode’s mouth. Nematoctonus geogenius can bedistinguished from N. angustatus by the pronounced con-striction in the hourglass of predatory adhesive knobs andby their conidia that germinate to first produce a swellingand then an adhesive hourglass cell. The Costa Rican iso-lates examined were derived from cultured fruiting bodiesof the teleomorph; this species was not seen or isolatedfrom any of the samples of substrates that were baited andstudied during this project. Isolates did not produce manyconidia and none of the Panagrellus bait nematodes werefound to be infected by the germinated conidia, as was ob-served by Thorn and Barron (1986) with isolates from On-tario and Iowa. In contrast, the predatory hourglass cellswere very efficient at adhering to the bait nematodes, andlarge numbers of adhesive cells subsequently formed aroundtheir carcasses.

Nematoctonus geogenius Thorn & G.L. Barron (1986)Figs. 3–4

Conidial pegs simple, 0.8–2.4 mm long � 0.8–1.6 mm wide.Predatory adhesive knobs predominantly intercalary; hour-glass cells 5.6–10.4 mm long � 2.4–5.6 mm wide at the base,with prominent median constriction; adhesive mucoid drop5–12 mm in diameter. Conidia cylindrical or fusoid, straight

754 Can. J. Bot. Vol. 85, 2007

# 2007 NRC Canada

or slightly curved near the tip, (4.8–)6.4–8.0(–9.6) mm � 1.6–2.4(–3.2) mm, Q = (2.3–)2.7–4.0(–4.5);forming an apical swelling prior to the germ tube; hour-glass cell 0.8–3.2 mm long � 0.8–1.6 mm, mucoid drop1.6–3.2 mm in diameter. Aleuriospores absent.

ISOLATES EXAMINED: Cultured once from Hohenbuehelia petalo-des collected in premontane wet forest, and once from infectednematodes in a sample from premontane rain forest (Table 1).One other collection of H. petalodes, not cultured, wasfound at ACG (Guanacaste), Guanacaste Prov., ParqueNacional Rincon de la Vieja, Santa Maria Biological Station,premontane wet forest, 10846’N 85818’W, 800 m (RGT960701/01).

SUBSTRATES: fallen trees with white rot, on buttress of brown-rotted huge fallen tree.

HABITAT: in primary forest and open secondary forest.

PREY: Panagrellus redivivus and unidentified Costa Ricannematodes.

TELEOMORPHS: Hohenbuehelia petalodes and H. tremula (thelatter not found in Costa Rica; Thorn and Barron 1986).

REMARKS: Nematoctonus geogenius is characterized by small,cylindrical to fusoid, straight or slightly curved conidia thatgerminate to produce a swelling followed by an hourglasscell surrounded by a mucoid drop and by predominantly in-tercalary large predatory hourglass cells surrounded by alarge adhesive mucoid drop. The predatory hourglass cellsattached to the nematode’s head and body and hyphae sub-sequently approached and entered the dead nematode’smouth. Unlike N. angustatus, bait and native soil nematodeswere infected by germinated conidia from soil and from cul-tures. Nematoctonus angustatus can be distinguished fromN. geogenius by its broader, less constricted hourglass cellsin predatory adhesive knobs and by its conidia that germinatewithout producing a swelling before producing an adhesivehourglass cell. The Costa Rican isolates studied are generallyconsistent with descriptions by Thorn and Barron (1986) ofisolates from Ontario, California, Europe, and Japan exceptfor shorter and narrower conidia in the Costa Rican isolates.Only two isolates of N. geogenius were obtained in CostaRica: one from a fruiting body of H. petalodes and theother from the sprinkle method. The isolate from the soilsprinkle plate occurred 5–10 d after sprinkling on two rep-licate plates, only infected a few nematodes, and was read-ily isolated into culture.

Nematoctonus leptosporus Drechsler (1943) Figs. 5–7Conidial pegs simple or commonly branched, 2.4–8 mm �

0.8–1.6 mm. Predatory adhesive knobs absent. Conidiahyaline, smooth, long, narrow and fusiform, (10.4–)17–24(–25.6) mm � 1.6–2.4 mm, Q = (6.0–)6.8–13.0(–15.5);forming hourglass cells 0.8–3.2 mm � 0.8–1.6 mm sur-rounded by a mucoid drop 0.8–4.0 mm in diameter at thetip of slender, sometimes bent germ tubes 0.8–4.8 mm �0.8 mm. Aleuriospores absent.

ISOLATES EXAMINED: Cultured once from a fruiting body of aHohenbuehelia sp. collected in Tropical Wet Forest, and re-corded three times from infected nematodes in samples fromLower Montane Moist Forest, Lower Montane Wet Forest,and Montane Rain Forest (Table 1).

SUBSTRATES: on dead hanging vines, aerial soil, and mosses, inhollow rotting logs, under dense shrubs, decaying bark, andleaf litter.

HABITAT: in primary forest and disturbed secondary forests.

PREY: Unknown for polyspore culture for teleomorph, andCosta Rican Bunonema sp. for isolates from organic debris.

TELEOMORPH: Unidentified species of Hohenbuehelia (poly-spore culture); unknown (organic debris isolates).

COMMENTS: Nematoctonus leptosporus is characterized by itsparasitoid habit, specificity to Bunonema prey, and by pro-duction of long fusoid conidia that germinate to produce anhourglass cell surrounded by a mucoid drop. This specieshas been recorded from decayed-leaf litter for deciduouswoods in Virginia (Drechsler 1943), as well as from NorthCarolina (Feder 1962), Queensland (McCulloch 1977), andNova Scotia (Alger 1980, although this record may representN. tylosporus lacking aleuriospores); in each of the subse-quent reports, this species was recorded just once in exten-sive surveys of nematophagous fungi. Conidia are longerand more slender than those of N. tylosporus, and no aleur-iospores are formed. Conidia of Costa Rican material areshorter than described by Drechsler (1943), 21–28 mm �1.7–2.2 mm, but their shape and the prey specificity fit thisspecies well. Germinated conidia from isolate RGT 010810/02 did not infect our bait nematodes (Panagrellus). This iso-late, from La Selva, fits this form-species best but may notrepresent the same biological species as those found insprinkle plates, attacking Bunonema in high-altitude sites.These, unfortunately, could not be cultured. All of the siteswhere this species was found were in perennially moist en-vironments.

Nematoctonus pachysporus Drechsler (1943) Figs. 8–10Conidial pegs simple, mostly 1.6–6.4 mm long � 0.8–

2.4 mm wide at the base. Predatory adhesive knobs absent.Conidia fusiform, (8.8–)12.0–16.8(–28.8) mm � (2.4–)3.2–4.8(–5.6) mm, Q = (2.14–)2.8–4.8(–6.5); producing a germtube 3.2–8.0(–17.6) mm � (0.8–)1.6–2.4 mm, with an apicaladhesive knob consisting of an hourglass cell 3.2–4.8 mmlong � 0.8–2.4 mm wide at the base surrounded by a mucoiddroplet 2.4–5.6 mm in diameter; the germ tube sometimesbranches to produce more than one hourglass cell. Aleurio-spores light yellow and coarsely warted when mature, hyalineand smooth or finely warted when young, ovoid, lenticular,to subglobose, (5.6–)8.8–12.8(–15.2) mm � (3.2–)4.8–7.2(–10.4) mm, Q = (1.0–)1.3–2.3(–2.7), abundant in someisolates but uncommon in others.

ISOLATES EXAMINED: cultured or recorded 17 times in samplesfrom tropical wet forest, premontane wet forest, premontanerain forest, lower montane moist forest, and lower montanewet forest (Table 1).

SUBSTRATES: in rich, organic soils, leaf litter or rotting wood,in terrestrial or epiphytic mosses.

HABITATS: in primary and secondary forests, in pastures andpotato fields.

PREY: Panagrellus redivivus and Rhabditis terricola (Thornand Barron 1986); Caenorhabditis elegans, P. redivivus andunidentified Costa Rican nematodes (this study).

Koziak et al. 755

# 2007 NRC Canada

TELEOMORPH: Hohenbuehelia izonetae Singer, not yet found inCosta Rica, Thorn and Barron 1986.

COMMENTS: Nematoctonus pachysporus is characterized by itsparasitoid behaviour, its warty, ovoid aleuriospores, and fu-soid conidia that produce hourglass cells surrounded by amucoid drop. Other parasitoid species with aleuriospores(e.g., N. tylosporus) can be distinguished from N. pachysporusby their narrower conidia, by their smaller and more finelywarted aleuriospores, and by their prey species. In the lab-oratory Nematoctonus pachysporus has a broader range ofprey species than does N. tylosporus, which has only beenobserved to consume Bunonema. (Drechsler 1941 and thisstudy). Nematoctonus pachysporus was originally discov-ered in nematode-infested tomato seedlings in Maryland(Drechsler 1943) and has since been reported in samplesof agricultural soils in England that were infested with po-tato root nematodes or cereal root nematodes (Duddington1954), in cultivated soils in Queensland, Australia (McCul-loch 1977), in Ontario farm soils (Barron 1978, Thorn andBarron 1986), and in leaf litter from farm soil in Tokyo,Japan (Saikawa and Arai 1986). The Costa Rican isolatesexamined are consistent with descriptions by Drechsler(1943), Saikawa and Arai (1986), and Thorn and Barron(1986). Nematoctonus pachysporus usually appeared after5–10 d of observation of sprinkle plates, but sometimespersisted among the last nematode-destroying fungi on theplates. Different isolates either consumed nematodes over theentire plate or consumed a select few nematodes, and mostisolates grew readily in culture. Isolates 04-RGTSN-526, 04-RGTSN-540, and 04-RGTSN-580 produce conidia with hya-line or light-yellow thicker cell walls, which are smooth orfinely warty and oval or ovoid in shape, (12.0–)12.8–15.2(–16.8) mm � 4.0–5.6 mm, Q = 2.3–3.6(–4.2). Theseconidia appear to be intermediates between aleuriospores andconidia, and were not observed to germinate or produce hour-glass cells. Nematoctonus pachysporus was the secondmostfrequently encountered Nematoctonus species during thisstudy. It was found mainly in premontane or lower montane

forests at middle elevations. Only isolate 01-RGTSN-600 wasfound at a lower elevation, in primary tropical wet forest atLa Selva. Persmark et al. (1995) found that nematodes weremore abundant in soils at higher elevations in Panama. Thissuggests that N. pachysporus is most abundant in perenniallymoist environments, where more nematodes may be foundfor consumption than occur in seasonally dry areas.

Nematoctonus robustus F.R. Jones (1964) Figs. 11–17Conidial pegs simple, 0.8–6.4 mm long � 0.8–1.6 mm

wide at the base. Predatory adhesive knobs predominantlyintercalary and occasionally terminal; hourglass cells 5.6–8.8 mm long � 2.4–3.2(–4.0) mm wide at the base, sur-rounded by an adhesive mucoid drop 5–10(–15) mm in di-ameter. Conidia kidney-shaped, sausage-shaped, or straightand cylindrical (4.8–)6.4–12.0(–16.0) mm � (1.6–)2.4–4.0(–6.4) mm, Q = (1.7–)2.3–4.0(–5.0), usually not germi-nating on WA with nematodes (for two exceptions, see Re-marks, below). Aleuriospores absent.ISOLATES EXAMINED: Cultured 9 times from fruiting bodies ofHohenbuehelia grisea collected in tropical wet forest, pre-montane moist forest (basal belt transition), premontane wetforest, premontane rain forest, and lower montane rain for-est, and isolated 9 times from infected nematodes in samplesfrom tropical wet forest, premontane wet forest, premontanerain forest, and montane wet forest (Table 1).SUBSTRATES: organic litter, rotting logs and wood, rich organicdebris, mosses, on fallen branches, and hanging vines.HABITAT: in primary and secondary forests and in clearings.PREY: Panagrellus redivivus and unidentified Costa Ricannematodes.TELEOMORPHS: Hohenbuehelia grisea and H. nigra (Costa Rica);H. approximans and H. cyphelliformis (not known fromCosta Rica; Thorn and Barron 1986). Ovrebo (1997) re-ported H. nigra from La Selva, but did not establish thatthe collection had an N. robustus anamorph.REMARKS: Nematoctonus robustus is characterized by cylindri-

Figs. 1–21. Figs. 1–2. Hohenbuehelia angustata (Nematoctonus angustatus state), both RGT 010802/04. Fig. 1. Germinated conidia withhourglass knobs surrounded by a mucoid drop (arrows), ungerminated conidia (arrowheads), and clamp connections (white arrows). Scalebar = 10 mm. Fig. 2. Predatory adhesive hourglass knobs (arrows) surrounded by mucoid drops, scale bar = 10 mm. Figs. 3–4.Nematoctonusgeogenius. Fig. 3. Hohenbuehelia petalodes (N. geogenius state), RGT 020615/16: conidia and fertile hyphae, scale bar = 5 mm. Fig. 4. 04-RGTSN-513: predatory hourglass knobs surrounded by mucoid drops on hyphae; inset, same from RGT 020615/16, scale bar = 5 mm. Figs.5–7. Nematoctonus leptosporus. Fig. 5. 04-RGTSN-543: conidium and germinated conidium with an adhesive knob (arrow) attached to abranched peg (arrowhead), scale bar = 5 mm. Fig. 6. 04-RGTSN-572: germinated conidia with long germ tubes and adhesive knobs (arrows),scale bar = 5 mm. Fig. 7. Hohenbuehelia sp. (Nematoctonus leptosporus state), RGT 010810/02: germinated and ungerminated conidia, scalebar = 5 mm. Figs. 8–10. N. pachysporus Fig. 8. 04-RGTSN-526: conidia (arrowheads) and germinated conidia with hourglass knobs surroundedby mucoid drops (arrows), one with two hourglass cells (asterisk); inset, 02-RGTSN-322: spiny aleuriospore, scale bar = 5 mm. Fig. 9. 04-RGTSN-574: conidium forming a germ tube (arrow) and aleuriospores (arrowheads) on pegs, scale bar = 5 mm. Fig. 10. 01-RGTSN-531: conidiaattached to pegs stemming from clamp connections (arrows), scale bar = 5 mm. Figs. 11–17. Nematoctonus robustus. Fig. 11. 04-RGTSN-535:curved conidia and a terminal predatory adhesive hourglass knob (arrow), scale bar = 5 mm. Fig. 12. 01-RGTSN-523: conidia of variousshapes, scale bar = 5 mm. Fig. 13. Hohenbuehelia grisea (N. robustus state), RGT 020623/05: intercalary predatory adhesive hourglass knobsurrounded by a mucoid drop (arrow), scale bar = 5 mm. Fig. 14. Hohenbuehelia grisea (N. robustus state), RGT 020620/07: conidia on pegsin whorls, scale bar = 5 mm. Fig. 15. 03-RGTSN-519: two predatory adhesive hourglass knobs and a straight conidium, scale bar = 5 mm.Fig. 16. 03-RGTSN-519: germinated conidia with hourglass knobs surrounded by mucoid drops (arrows), scale bar = 5 mm. Fig. 17. 03-RGTSN-519: variously curved conidia, scale bar = 10 mm. Figs. 18–21. Nematoctonus tylosporus. Fig. 18. 04-RGTSN-579: fertile hyphae(arrows) emerging Bunonema sp. with branched pegs (arrowheads) surrounded by germinated conidia with adhesive knobs, scale bar = 25 mm.Figs. 19 and 20. 04-RGTSN-562: aleuriospores (arrows), fertile hyphae with clamp connections, conidiogenous peg (white arrow), and ger-minated conidia with adhesive knobs (arrowheads), scale bars = 5 mm. Fig. 21. 04-RGTSN-512: abundant conidiation on WA with nema-todes; some conidia germinated to form adhesive knobs (arrowheads), scale bar = 10 mm.

756 Can. J. Bot. Vol. 85, 2007

# 2007 NRC Canada

Koziak et al. 757

# 2007 NRC Canada

cal and usually curved conidia and predominantly interca-lary predatory hourglass cells surrounded by an adhesivemucoid drop. Predatory hourglass cells were observed to at-tach to the tail, body, and mouth of nematodes, and hy-phae were observed to approach and penetrate the deadnematode’s body directly or to enter openings, such as themouth and anus. What appears to be this form-species, ori-ginally discovered in leaf litter collected from a forest re-serve in Ghana, Africa (Jones 1964), has been reportedfrom saunas in Finland (Salonen and Ruokola 1968), fromsheep and cattle yards in New Zealand (Fowler 1970),from woodland sites in Iowa (Kitz and Embree 1979),from chicken manure in Nova Scotia (Alger 1980), fromfarm and woodland soils in Ontario and Quebec (Thornand Barron 1986), from rotten wood in Taiwan (Liou andTzean 1997), and from sheep dung in Brazil (Saumell etal. 2000). The form-species Nematoctonus robustus canalso be cultured from any of its several teleomorph speciesfound fruiting on rotting wood. The Costa Rican isolatesexamined are consistent with descriptions by Jones (1964)and Thorn and Barron (1986). Isolates found by the sprinkle-plate method usually appeared after 5–10 d of observation.Nematoctonus robustus was usually the first nematode-destroying fungus to appear on the plates, but isolateswere usually restricted to a small area of the plate andwere not found on all of the replicate plates. All isolatesgrew readily in culture from washed, infected nematodes.Isolates RGT 040611/01 (cultured from a fruit body) andmonokaryon 03-RGTSN-519 (from soil) produced conidiathat germinated on WA with nematodes. In the former,these conidia were generally plump, cylindrical, and straightor slightly curved, 7.2–11.2 mm � 3.2–4.8 mm, Q = 2.0–2.8,and produced germ tubes 2.4–18.4 mm long and 1.6–2.4 mm wide without forming adhesive knobs. In the latter,the germ tube formed one or more hourglass cells 3.2–5.6 mm long by 0.8–3.2 mm, each surrounded by a mucoiddrop 3.2–8.0 mm � 3.2–6.4 mm. Germinating conidia havenot been reported previously in this species. Nematoctonusrobustus was the most frequent species found during thisstudy and was found in seasonally dry and perenniallymoist environments, at low and high elevations. The broadecological amplitude together with the morphological varia-bility among isolates and multiple teleomorph species sug-gests that several biological species may be combinedunder this form-species.

Nematoctonus tylosporus Drechsler (1941) Figs. 18–21Conidial pegs simple or occasionally branched, (1.6–)4.0–

12.0(–15.2) mm long � 0.8–3.2(–5.6) mm wide at the base.Predatory adhesive knobs absent. Conidia narrow, fusiform,(8–)11.2–18.4(–21.6) mm � 1.6–3.2(–4.0) mm, Q = (4.3–)4.7–10.0(–11.5); producing germ tubes 0.8–10 mm � 0.8 mm,usually with an apical adhesive knob 0.8–3.2(–4.0) mm �0.8–1.6 mm surrounded by a mucoid drop 0.8–2.4 mm �0.8–2.4 mm; differentiated adhesive knobs were not alwaysobserved at the tips of long tapering germ tubes. Aleuriosporeslight yellow, finely warted or smooth, ovoid or almond-shaped, 5.6–10.4(–11.2) mm � (1.6–)2.4–4.0(–4.8) mm, Q =(1.5–)1.8–3.5(–4.0), uncommon in most isolates and appa-rently absent in others.

ISOLATES EXAMINED: Cultured or recorded 12 times from in-

fected nematodes in samples from premontane rain forest,lower montane wet forest, montane wet forest, and montanerain forest (Table 1).

SUBSTRATES: rich, organic soil and rotted wood, mossy soil,and leaf litter.

HABITATS: in primary and secondary forests, shrubby fields,bamboo forest, and pastures.

PREY: Unidentified Costa Rican Bunonema sp.

TELEOMORPH: None known.

REMARKS: Nematoctonus tylosporus is characterized by itsparasitoid behaviour, its finely warted or smooth, ovoid oralmond-shaped aleuriospores, and narrow and fusoid coni-dia. Other parasitoid species with aleuriospores (e.g.,N. pachysporus) can be distinguished from N. tylosporus bytheir broader conidia, by their larger and more coarselywarted aleuriospores, and by their prey species. Nematocto-nus tylosporus has only been observed to consume speciesof Bunonema (Drechsler 1941, this study). Nematoctonuspachysporus has a broader range of prey than this, as it isable to consume Caenorhabditis elegans, Panagrellus redi-vivus, Rhabditis terricola (Thorn and Barron 1986), and un-identified Costa Rican nematodes in this study. The CostaRican isolates examined are consistent with descriptions byDrechsler (1941), who noted that aleuriospores were pro-duced ‘‘occasionally’’ relative to the smooth, thin-walledconidia. In five of our records, aleuriospores were producedsparingly, and in seven others, they were not seen. The iden-tical conidial morphology, prey, and habitats of these latterisolates all suggest conspecificity with N. tylosporus. Nema-toctonus tylosporus was originally discovered in leaf litterfrom Washington D.C. (Drechsler 1941) and has since beenreported from soil in Nova Scotia (Alger 1980), from wood-land sites in Iowa (Kitz and Embree 1979), from mosses androtting leaves and wood in North Carolina (Feder 1962),from mosses in England (Duddington 1951), from leaf litterin Denmark (Shepherd 1956), and from soil near red cloverin Iowa (Norton 1962). Isolates found by the sprinkle-platemethod usually appeared after approximately 10–14 d of ob-servation, approximately 5–10 d after Bunonema flourishedon the WA plates. Isolates usually consumed Bunonemaover the entire plate, but others consumed only a few nem-atodes. Nematoctonus tylosporus did not frequently reappearon the replicate plates. Twelve samples yielded Nematocto-nus tylosporus during this study, but only 04-RGTSN-512from Guayabo National Monument could be cultured; theothers failed to grow from infected nematodes transferred toAntPDA or MEB. It was found in wet habitats at mid tohigh elevations in organic soils or well-rotted wood. Thissuggests that N. tylosporus occurs in perennially moist envi-ronments where Bunonema nematodes may be more com-mon than in seasonally dry areas.

DiscussionIn this first report of Nematoctonus from Costa Rica, we

found six morphospecies, all previously described, and allreported from temperate areas in the United States and Can-ada (Drechsler 1941, 1943; Thorn and Barron 1986). Thornand Barron (1986) reported eight species of Nematoctonusfrom Ontario, Canada. They recovered five species of Nem-

758 Can. J. Bot. Vol. 85, 2007

# 2007 NRC Canada

atoctonus from 713 samples of soil and organic debris(N. geogenius, N. hamatus, N. leiosporus, N. subreniformis,and N. robustus), confirmed the report by Barron (1978) ofN. pachysporus that had been isolated from soil by theBaermann funnel and centrifugation techniques, and isolatedtwo additional species from Hohenbuehelia fruiting bodies(N. angustatus and N. brevisporus). In their study, 5 isolatesof four species were recovered via soil sprinkling, 29 iso-lates of three species were recovered using the Baermannfunnel method, and 21 isolates of five species were recov-ered using a centrifuge technique (Thorn and Barron 1986).Our samples of soil and organic debris from Costa Ricayielded few nematodes, so we discontinued the use of theBaermann funnel technique for these samples in 2001, andthe centrifugation technique was not attempted. Nonetheless,we detected most of the species reported from North Amer-ica. The apparent lack of endemic species in this country ofgreat biodiversity supports the contention that many nema-tophagous fungi are widely distributed (Barron 1978).

Our samples were obtained from 6 of the 11 conservationareas in Costa Rica, to the exclusion of the extreme north-east and southeast of the country. Not sampled were theA.C. La Amistad Caribe, A.C. Arenal-Huetar Norte, A.C.Marina Isla del Coco, A.C. Tempisque, or A.C. Tortuguero.Our sampling of the 12 Holdridge life zones in Costa Ricawas unequal (Table 1), based primarily on their geographicextent and ease of access. Particularly under-sampled werethe premontane moist forest, in Costa Rica largely convertedto agriculture and urban areas such as Alajuela, Cartago,Heredia, and San Ramon; the lower montane moist forestand subalpine rain paramo, of very limited extent in CostaRica; and the lower montane rain forest, montane wet for-est, and montane rain forest, of high elevation and difficultaccess. Our survey concentrated on natural habitats protectedin national parks and conservation reserves and includedfew samples of agricultural soils, particularly barnyard soilor compost, which were very productive for Nematoctonusin surveys by Barron (1978) and Thorn and Barron (1986).Future surveys of nematode-destroying fungi in Costa Ricamight benefit from sampling these other habitats, especiallyin areas where cool temperatures retard microbial decom-position of these highly organic substrates.

The nematodes used as bait in our soil sprinkle plateswere either Panagrellus (order Rhabditida, suborder Cepha-lobina) or Caenorhabditis (order Rhabditida, suborder Rhab-ditina), although at least some of the native nematodes inthe samples eventually reproduced on the sprinkle platesand acted as a more natural and diverse bait. Nonetheless,greater diversity of nematode-destroying fungi might havebeen detected with the use of a wider assortment of baitnematodes, particularly Bunonema. Our attempts to cultureBunonema for use as bait were unsuccessful. When Buno-nema nematodes were gently washed or transferred fromtheir original water agar plate on a piece of agar to freshwater agar plates, they never recovered. The transport andstorage of soil and organic debris samples prior to process-ing might also have reduced the viability of some Nematoc-tonus species or their nematode prey. Future surveys couldfocus on specific areas of Costa Rica, processing small num-bers of samples locally without delay, to obtain a more com-plete estimate of Nematoctonus diversity in Costa Rica.

The best substrates for Nematoctonus were moss, organicsoil, decaying leaf litter, and rotting wood. Samples from theLas Alturas and Santa Maria Biological Stations (premon-tane wet forest), Guayabo National Monument (premontanerain forest), Reserva Santa Elena and Volcan Barva (lowermontane wet forest) yielded three species of Nematoctonus,and nine other sites yielded two species (Table 1). In gen-eral, species of Nematoctonus were found most frequentlyin cooler, moist or wet habitats at higher elevations. This re-sult supports findings from Persmark et al. (1995), wherenematodes and nematode-destroying fungi were found ingreater frequencies and densities at cooler and higher eleva-tions. Cooler habitats at higher elevations may be more fa-vourable to nematode populations because of possibledecreased competition for energy and resources, as com-pared with lower elevation tropical habitats, where competi-tion by other members of the soil microbial and microfaunalcommunities is greater (Fittkau and Klinge 1973; Procter1984). Their organic substrates may also be less ephemeraland subject to rapid turnover in perennially wet or cool hab-itats, allowing for a build-up of greater nematode popula-tions and communities. This, more than the dryness of thedry season, may explain why no isolates of Nematoctonuswere obtained from soils collected in the seasonally dry for-ests of Guanacaste Province (tropical dry forest and premon-tane moist forest basal belt transition) nor from disturbedand seasonal habitats in the tropical moist forest (Table 1).Hohenbuehelia grisea, the teleomorph of N. robustus, wascollected on woody substrates in the dry forest areas of Gua-nacaste (at Los Almendros and Santa Rosa), but its ana-morph was not recovered in sprinkle plates of organicmaterials collected in the same areas. It must be there, butit may be restricted to woody substrates that are less proneto drying out than are the local soils and litter.

It appears that parasitoid species of Nematoctonus are ca-pable of producing sexual fruiting bodies, in contrast to pre-vious predictions that parasitoids had lost this ability(Barron and Dierkes 1977; Thorn and Barron 1986). Cul-tures identified as the parasitoid species N. cylindrosporus,N. pachysporus (Thorn and Barron 1986), and N. leptospo-rus (this study) were isolated from fruiting bodies of Ho-henbuehelia. As first suggested by Drechsler (1949), hugenumbers of microscopic individual colonies (ramets), eacharising from a single infected nematode, must unite throughanastomosis to muster the nutrients required for productionof a macroscopic fruiting body. Alternatively, fruiting bodyproduction in these species may use resources gathered by asaprotrophic mycelium feeding on lignocellulosic wastes —a mycelium that we have not been able to detect in oursampling, while nematodes are captured by a dispersed con-idial phase, which we detect in our soil sprinkle plates. Thiscould explain the apparent conundrum in both parasitoidand predatory species of teleomorphs being found on trunks,branches, and hanging vines, whereas anamorphs are de-tected in soil, leaf litter, and mosses. These and other ques-tions of the biology of Nematoctonus remain for furtherstudy.

AcknowledgementsThis research was supported by funding from the

U.S. National Science Foundation (NSF DEB-0072756 to

Koziak et al. 759

# 2007 NRC Canada

R.G. Thorn and G.K. Brown, University of Wyoming), theNatural Sciences and Engineering Research Council ofCanada (DG 238464–01 to R.G. Thorn), and the CostaRica – USA Foundation (01–01-CT to F. Chavarria), andby logistical support from INBio and ACG. We thank EddyCambronero, Ana Martinez, Adelina Morales, DanielaTorres, all of ACG, and Claire Tuason (UWO) for assistancein sampling; Michelle Marcus, Gladys Mok, Ruby Sadera,and Diana Whellams (UWO) for assistance with isolationand maintenance of cultures; and Ian Craig and Rick Harris(UWO) for help with digital imaging and microscopy. Weare grateful for logistical support in Costa Rica fromDr. Luis Diego Gomez (Las Cruces, San Vito de Java),Manrique Gonzales (CATIE, Turrialba), Isaac Lopez(INBio, Bijagua), Dr. Jorge Sandoval (CORBANA,Guapiles), Paulino Valverde (Sirena, Corcovado NationalPark), and numerous other field station managers and staff;and to Alvaro Herrera Villalobos (INBio) for obtainingcollecting and export permits for this research from theCosta Rican Ministerio del Ambiente y Energıa (MINAE).

ReferencesAlberto, E., Fazio, A., and Wright, J.E. 1998. Reevaluation of

Hohenbuehelia nigra and species with close affinities.Mycologia, 90: 142–150. doi:10.2307/3761024.

Alger, M.E. 1980. Nematophagous fungi of Kings County, NovaScotia. M.Sc. thesis, Department of Biology, Acadia University,Wolfville, N.S.

Bailey, F., and Gray, N.F. 1989. The comparison of isolationtechniques for nematophagous fungi from soil. Ann. Appl. Biol.114: 125–132.

Barron, G.L. 1977. The nematode-destroying fungi. CanadianBiological Publications, Guelph, Ont.

Barron, G.L. 1978. Nematophagous fungi: endoparasites ofRhabditis terricola. Microb. Ecol. 4: 157–163.

Barron, G.L., and Dierkes, Y. 1977. Nematophagous fungi:Hohenbuehelia, the perfect state of Nematoctonus. Can. J. Bot.55: 3045–3062.

Bucaro, R.D. 1983. Hongos Nematofagos de El Salvador. Rev.Biol. Trop. 31: 25–28.

Dalla Pria, M., Ferraz, S., and Muchovej, J.J. 1991. Isolamento eidenticacao de fungos nematofagos de amostras de solo dediversas regiones do Brasil. Nematol. Bras. 15: 170–176.

Dias, W.P., Ferraz, S., and Muchovej, J.J. 1995. Deteccao,isolamento e identicacano de fungos predadores de nematoidesem amostras de solo de diferentes regiones do Brasil. Rev.Ceres, 42: 615–620.

Drechsler, C. 1941. Some hyphomycetes parasitic on free-livingterricolous nematodes. Phytopathology, 31: 773–802.

Drechsler, C. 1943. Two new basidiomycetous fungi parasitic onnematodes. J. Wash. Acad. Sci. 33: 183–189.

Drechsler, C. 1946. A clamp-bearing fungus parasitic and predac-eous on nematodes. Mycologia, 38: 1–23. doi:10.2307/3755043.

Drechsler, C. 1949. A nematode-capturing fungus with anastomosingclamp-bearing hyphae. Mycologia, 41: 369–387. doi:10.2307/3755230.

Drechsler, C. 1954. A nematode-capturing fungus with clamp-con-nections and curved conidia. J. Wash. Acad. Sci. 44: 82–85.

Duddington, C.L. 1951. The ecology of predaceous fungi: prelimin-ary survey. Trans. Br. Mycol. Soc. 34: 322–341.

Duddington, C.L. 1954. Nematode-destroying fungi in agriculturalsoils. Nature (London), 173: 500–501. doi:10.1038/173500a0.PMID:13144793.

Durschner-Pelz, U.U. 1987. Traps of Nematoctonus leiosporus —an unusual feature of an endoparasitic nematophagus fungus.Trans. Br. Mycol. Soc. 88: 129–130.

Fazio, A., and Alberto, E. 2001. Two new species of Hohenbuehe-lia from Argentina. Mycotaxon, 77: 117–125.

Feder, W.A. 1962. Nematophagus fungi recovered around High-lands, North Carolina. Plant Dis. Rep. 46: 872–873.

Fittkau, E.J., and Klinge, H. 1973. On biomass and trophic struc-ture of the central Amazonian rain forest ecosystem. Biotropica,5: 2–14. doi:10.2307/2989676.

Fowler, M. 1970. New Zealand predacious fungi. N.Z. J. Bot. 8:283–302.

Gamundi, I.J., and Spinedi, H.A. 1982. Sobre la presencia de hon-gos depredadores de nematodos en la Argentina. Physis (BAires), 41: 37–46.

Gazzano, S. 1973. Hongos depredadores de nematodos libres delUruguay. Rev. Biol. Urug. 1: 191–205.

Giuma, A.Y., and Cooke, R.C. 1971. Nematotoxin production byNematoctonus haptocladus and N. concurrens. Trans. Br. Mycol.Soc. 56: 89–94.

Giuma, A.Y., and Cooke, R.C. 1972. Some endozoic fungi parasiticon soil nematodes. Trans. Br. Mycol. Soc. 59: 213–218.

Godfray, H.C.J. 1994. Parasitoids: behavioral and evolutionaryecology. Princeton University Press, Princeton, N.J.

Gray, N.F. 1984. Ecology of nematophagous fungi: comparison ofthe soil sprinkling method with the Baermann funnel techniquein the isolation of endoparasites. Soil Biol. Biochem. 16: 81–83.doi:10.1016/0038-0717(84)90131-7.

Gray, N.F., and Duff, K.R. 1982. Some preliminary observationson predaceous fungi from Ireland. Ir. Nat. J. 20: 378–380.

Holdridge, L.R. 1971. Forest environments in tropical life zones; apilot study. Pergamon Press, New York.

Jaffee, B.A., Strong, D.R., and Muldoon, A.E. 1996. Nematode-trapping fungi of a natural shrubland: tests for food chain invol-vement. Mycologia, 88: 554–564. doi:10.2307/3761149.

Janzen, D.H. 1977. What are dandelions and aphids? Am. Nat. 111:586–589. doi:10.1086/283186.

Janzen, D.H. (Editor). 1983. Costa Rican natural history. Univer-sity of Chicago Press, Chicago, Ill.

Jones, F.R. 1964. Nematoctonus robustus sp. nov. Trans. Br. My-col. Soc. 47: 57–60.

Kirk, P.M., Cannon, P.F., David, J.C., and Stalpers, J.A. 2001. Dic-tionary of the Fungi, 9th ed. CAB International, Wallingford, UK.

Kitz, D.J., and Embree, R.W. 1979. Nematode-destroying fungi ofJohnson County, Iowa. Proc. Iowa Acad. Sci. 86: 19–21.

Lappe, P., and Ulloa, M. 1983. Nematode destroying fungi in somesoils of Mexico. Cultivation and identification of depredatorsand prey. Bul. Soc. Mex. Micol. 17: 99–113.

Liou, G.Y., and Tzean, S.S. 1997. Phylogeny of the genus Arthro-botrys and allied nematode-trapping fungi based on rDNA se-quences. Mycologia, 86: 876–884.

Lyman, G.R. 1907. Culture studies on polymorphism of Hymeno-mycetes. Proc. Boston Soc. Nat. Hist. 33: 125–209.

Mahoney, C.J., and Strongman, D.B. 1994. Nematophagous fungifrom cattle manure in four states of decomposition at three sitesin Nova Scotia, Canada. Mycologia, 86: 371–375. doi:10.2307/3760567.

McCulloch, J.S. 1977. A survey of nematophagous fungi inQueensland. Qld. J. Agric. Anim. Sci. 34: 25–34.

Morgan-Jones, G., Rodriguez-Kabana, R., and Tovar, J.G. 1984.Fungi associated with cysts of Heterodera glycines in the CaucaValley, Colombia. Nematropica, 14: 173–177.

Naves, R.L., and Campos, V.P. 1991. Ocorrencia de fungos preda-dores de nematoides no sul de Minas Gerais e estudo da capaci-

760 Can. J. Bot. Vol. 85, 2007

# 2007 NRC Canada

dade predatoria e crescimento in vitro de alguns de seus isola-dos. Nematol. Bras. 15: 152–162.

Nobles, M.K. 1937. Production of conidia by Corticium incrustans.Mycologia, 29: 557–566. doi:10.2307/3754507.

Norton, D.C. 1962. Iowa fungi parasitic on nematodes. Iowa Acad.Sci. 69: 108–117.

Omar, M.B., Bolland, L., and Heather, W.A. 1979. A permanentmounting media for fungi. Bull. Br. Mycol. Soc. 13: 31–32.

Ovrebo, C.L. 1997. The agaric flora (Agaricales) of La Selva Bio-logical Station, Costa Rica. Rev. Biol. Trop. 44 (Suppl. 4): 39–57. PMID:9404514.

Persmark, L., Marban-Mendoza, N., and Jansson, H.-B. 1995. Ne-matophagous fungi from agricultural soils of Central America.Nematropica, 25: 117–124.

Procter, D.L.C. 1984. Towards a biogeography of free-living soilnematodes. I. Changing species richness, diversity and densitieswith changing latitude. J. Biogeogr. 11: 103–117. doi:10.2307/2844684.

Rubner, A. 1994. Predacious fungi from Ecuador. Mycotaxon, 51:143–151.

Ruokola, A.-L., and Salonen, A. 1967. On nematode-destroyingfungi in Finland. J. Sci. Agric. Soc. Finl. 39: 119–130.

Saikawa, M., and Arai, K. 1986. Electron microscopy on the nema-

tode-endoparasitic fungus Nematoctonus pachysporus. Trans.Mycol. Soc. Jpn. 27: 33–39.

Salonen, A., and Ruokola, A.-L. 1968. On nematode-destroyingfungi in Finland II. J. Sci. Agr. Soc. 40: 142–145.

Santos, M.A., Ferraz, S., and Muchovej, J.J. 1991. Detection andecology of nematophagous fungi from Brazilian soils. Nematol.Bras. 15: 121–134.

Saumell, C.A., Padilha, T., and Santos, C.D.P. 2000. Nematopha-gous fungi in sheep faeces in Minas Gerais, Brazil. Mycol. Res.104: 1005–1008. doi:10.1017/S095375629900218X.

Shepherd, A.M. 1956. A short survey of Danish nematophagousfungi. Friesia, 5: 396–408.

Singer, R. 1989. New taxa and new combinations of Agaricales(Diagnoses fungorum novorum Agaricalium IV). Fieldiana Bot.21: 1–133.

Stadler, M., Sheldrick, W.S., Dasen-Brock, J., Steglich, W., andAnke, H. 1994. Antibiotics from the nematode-trapping basidio-mycete Nematoctonus robustus. Nat. Prod. Lett. 4: 209–216.

Thorn, R.G., and Barron, G.L. 1986. Nematoctonus and the TribeResupinateae in Ontario Canada. Mycotaxon, 25: 321–454.

Vandendries, R. 1934. La polarite sexuelle et le regime conidien.Bull. Soc. Mycol. Fr. 50: 202–212.

Koziak et al. 761

# 2007 NRC Canada