Rev. Biol. Trop., 45(4): 1349-1383, 1997

Costa Rican gut fungi (Trichomycetes) imeeting lotic inseet larvae

Robert W. Lichtwardt Departmentof Botany, University of Kansas,Lawrence, KS 66045-2106, U.S.A.

Received 18-IV-1997. Corrected 29-V-1997. Accepted 28-VI-1997.

Abstract: Trichomycetes infecting freshwater Ephemeroptera and Díptera (Siínuliidae, Chironomidae) in various life zones oC Cósta Rica were studied. Insect populations in 36 streams were sampled, a few repeatedly, at four different

. perlods of time over seven years. Eleven new species of Harpellales are reported, plus six species knownto occur also in nontropical parts oí the world. Included are new species of Harpella and Pennella, a new monotypic genus (GraminelloidesJ, and four new species of the previously monotypic genera Spartiella and Genistellospora. Seven new Smittiúm species, including three that are not named at this time, were cultured axenically. A new Amoebidium (Amoebidiales) was found in a stream polluted with organic matter. In the same polluted stream more species of dipteran larvae and a greater number of gut fungi were present than in stretclies of unpolluted water upstream. The overall diversity of Harpellales and their aquatic insect hosts in most Costa Rican streams appeared to be lower than what the author has found in many lotic habitats in more northem and southem regions of the world, and thus this orderof fungi may be an exception to the 'latitudinal species diversity gradient' concept as it applies to many other tropical organisms. A key to all 23 Trichomycetes now known to be present in Costa Rica is provided.

Keywords: Aquatic insects. Diptera, Ephemeroptera, symbiosis, Trichomycetes, tropics.

Costa Rica has numerous types of freshwa­ published investigation of the prevalence and ter insect larvae whose hindguts or midguts are distribution of gut fungi in tropical str.eams. infected with HarpeHales, one of several orders Harpellales found in Costa Rican streams of the cosmopolitan arthropod-associated fun­ incIude one new genus and 11 new species, as gal Class Trichomycetes (Zygomycota) well as six species that have been previously (Lichtwardt 1986, 1996). Larval insect hosts of reported from nontropical regions of the world. Harpellales are Ephemeroptera (mayflies), Additionally, three possibly new species of Plecoptera (stoneflies), and many families of Smittium are described and illustrated, but not Nematocera (lower Diptera), including named at this time. AH seven lotic species of Simulüdae (blackflies), Chironomidae (nonbit­ Smittium were cultured axenical1y. Also found ing midges), Culicidae (mosquit oes), in Costa Rica were two genera and one new Ceratopogonidae (biting midges), Tipulidae species of Amoebidiales. Amoebidiale.s, previ­ (craneflies), and other families of dipterans not ously c1assified among Trichomycetes, are pro­ collected in Costa Rica. A previous publica­ tists of unknown phylogenetic affinities, and are tion (Lichtwardt, 1994) described harpellid gut believed not to be fungi (Lichtwardt 1986). fungi in Costa Rican insects that live in phy­ Rather, they constitute a convergent group of totelms, especially bromeliads, and other lentic organisms that oftenlive in or on thesame kinds habitats such as swamps; The current publica­ of larval hosts inhabited by harpellid gut fungí. tion is a study of Harpellales ibat live in stream A final, future paper in this series on Costa insects, representing a broader range of hosts Rican Trichomycetes will deal with the other and fungal species. This constitutes the first two orders, Asellariales and Eccrinales, associat- 1350 REVISTA DE BIOLOGIA TROPICAL ed with marine and terrestrialmature artJtropods. stage of fungal development. Most Trichomycetes appear to be harrnless commensals during their life within the arthro­ pod gut, or may at times benefit their hosts by MATERIALS AND METHODS providing sorne level of organic nutrients when the arthropods are nutritionally impoverished Collections of Costa Rican lotic insects (Hom and Lichtwardt 1981). An exception is were made in 1984 from 18 September to 25 Smittium morbosum Swee�ey that usually kills November, in 1986 from 3 June to 21 July, in the mosquito larvae within which it lives 1988 from 21 May to 28 June, and in 1991 (Sweeney 1981; Sato et al. 1989; López Lastra from 13 October to 22 November. During each 1990). It is now known that sorne species of of those years collections were made at the La Harpellales on occasion grow into the ovaries Selva Biological Station (Organization for of maturing insect hosts where fungal cysts Tropical Studies) and in and near the develop (Moss and Descals 1986; Labeyrie et Monteverde Cloud Forest Reserve (Tropical al. 1996). This results in sterilization of the Science Center). ConsequentIy, most of the adult female who then disperses the fungus streams in those areas were used repeatedly as when she "oviposits" the cysts within other sources of specimens. Many of the sites in breeding sites. Since all Harpellaleslive within Guanacaste Province were also visÍted more the nonflying larval stages of aquatic insects, it than once in 1988 and 1991. Streams in other has been speculated that possibly most or all of parts of Costa Rica were used in most cases for these fungi will be found to have this same single sets of collections. Table 1 lists the gen­ basic dispersion mechanism (Lichtwardt 1996). eral regions, life zones, and streams where col­ Thus, whether Harpellales infect or irifest their lections were made. Figure 1 illustrates the insect hosts may depend upon the particular distribution of study sites in Costa Rica.

TABLE 1 Regions and streams in Costa Rica where trichomycete-infected lotic insects were collected between 1984 and 1991

Location and general life zone Stream sites1

La Selva Biological Station Quebrada El Salto (ahove waterfall, or at Camino (Iow wet foresO Central, or at Camino Circular Lejano) (1); Q. Esquina (2); Q. Sábalo (3); Q. Surá (4); Q. El Taconazo(5)

Monteverde Q. Cuecha (upstream Río Guacimal) (6); R. Guacimal (c1oud forest, lower montane rain (7); Q. Máquina(8); within Reserve: headwaters of foresO severa! streams along SenderoBosque Nuboso, El Camino, and near campground (9)

Orosi valley, SE of Cartago R. Macho (10); R. Grande de Orosi (11); R. Chirí (12) (Iower and premontane moist foresO

NW of Heredia, Rt. 9 (Iower montanerain forest) R. Chorreras (13); R. Quizarraces (14)

SW of San José R. Tabarcia (15); Q. Chorrodel Padre (16); unnamed (premontane wet forest) streamS of Q. Chorrodel Padre (17)

Cordillera de Talamanca, on both S fork R. Humo (18); R. Parrita Chiquito (19); W branch sides of the divide (montane wet Q. Macho Mora (20); several unnamed streamsnear Rt. 2 and moist forest) (21)

Vicinity of Cahuita and Puerto Q. Matarrita (22); Q. Hotel (23) Viejo (coastal moist forest)

SE of Quepos R. Portalón (24); severa! unnamed streamsE of R. (coastal wet forest) Savegre,E of Matapalo,and near road lo Portalón (25)

Golfito(coastal wet forest) Cataratain Golfilo Reserve(26) ( Continued... ) Lichtwardt: Costa Rican gut fungi (Trichomycetes) 1351

South oC Las Cruces E branch R. Jaba, SnakeCreek, Lutite Spring, (premontane wet Corest) The Springon Water Trail (Wilson Botanical Garden) (27); Q. Cantarrana (28); R. Caracol (29)

Guanacaste Province (dry forest) R. Piedras (30); R. Corobicí (31); R. Animas (32); small stream in Santa Rosa N.P. (33); R. Tenorio (34)

Cordillera de Guanacaste (premontane wet forest) R. Bijagua (35); R. Naranjo (36)

I BoldCace numbers are site locations reCerencedin the text. Also refer to Fig. l.

such mounts; all types are deposited at the Farlow Herbarium (FH), Harvard University. Voucher specimens of insects, either dissected or undissected, were preserved in 70% ethanol for later identification by specialists whose names are given in the Acknowledgment sec­ tion. Any relevant pupae or adults collected in the field or that developed in the laboratory were also preserved. Axenic cultures of Harpellales were suc­ cessfulIy obtained fromsorne of many isolation attempts, using procedures that have been pub­ lished (Lichtwardt 1986). For culture attempts, fungal specimens in fue removed hindgut of an insect were kept as undisturbed as possible, usualIy after inspection with a compound microscope, washed at least twice in distilled Fig. 1. Map of Costa Rica showing collection sites. Small dots represent oneto three streams; largerdots representgen­ water with an added penicillin-streptomycin eral locations oC Cour or more stream sites. Refer to Table 1 mixture, then transferred to 60-Jlm petri dishes Cor more specificdata. containing 1/10 BHIv agar medium [dilute Difco brain-heart infusion (0.37 g per 100 mI Insect larvae were obtained from rocks, distilled water) with vitamins (thiamine, sticks, vegetation, and various other kinds of biotin»), to which a thin layer of sterile distilled substrates in the water to which lotic insects water and antibiotics were added afterthe 1.5% cling. Where appropriate, stream bottoms were agar had gelled. Successful cultures were disturbed, and the released insects were cap­ transferred to agar slants of 1/10 BHIv contain­ tured with a neto They were placed in small ing a small amount of sterile distilled water, jars containing mÍnimal amounts of water, and and were refrigerated after growth had in most instances kept on ice to ensure their occurred. Representative isolates of new live transport to a laboratory, where they were species will be deposited with the American maintained in a refrigerator until dissected. Type Culture Collection, Rockville, Maryland, Removal of,hindguts and midguts were done U.S.A. under a dissecting microscope using magnifica­ The isozyme patterns of Smittium spp. iso­ tions between 9 and 54X, and living fungal lated in Costa Rica used in this report are part material was studied in slide water mounts with of an earlier data matrix that formed the basis a phase-contrast compound microscope. of a more comprehensive investigation of cul­ Photographs of living gut fungi were made on tured Trichomycetes (Grigg 1994; Grigg and color transparency film with a photomicro­ Lichtwardt 1996). Isozymic relationships in the graphic unit equipped with electronic flash. current study used numerical taxonomy and Specimens were preserved on slides by infil­ multivariate analysis systems (NTSYS-pc ver. trating lactophenol cotton-blue under the cov­ 1.80) (Rohlf 1993) with unpaired group mean erslip, then sealing the coverslip to the slide averages (UPGMA), and were computed from with clearfinge rnail polish. Most type speci­ sequential, agglomerative, hierarchical, and mens of the new taxa published here consist of nested (SAHN) clustering methods. 1352 REVISTA DE BIOLOGIA TROPICAL

DESCRIPTIONS OF NEWFUNGAL diametro subaequante. Trichosporae sig­ SPECIES moideae, rarius curvulae vel subconspiratae, (98-)130(-140) x 3-4 f.!m, appendiculis 2-4. Table 2 pro vides a list of new taxa Zygosporae ignotae. In larvarum Simuliidarum described in this paper, together with other membrane peritrophico. Typus: FH (CR-252-2). species of Trichomycetes currently known to Mature thalli 180-380 x 6-9 f.!m, normally infect Costa Rican aquatic insects. with about 8 generative cells but ranging from 4-46 per thallus. Base rounded with a holdfast Harpella tica Lichtw., sp. nov. about same diameter as the thallus. (Figs. 2-6) Trichospores sigmoid, more rarely curved to almost coiled, (98-) 130(-140) x 3-4 11m, with 2 Thallimaturi 180-380 x 6-9 f.!m, vulgo cellu­ or 4 appendages. Zygospores unknown. On las genitalescirca 8, sed nonnumquam 4-46 ger­ peritrophic membrane of Simuliidae larvae. entes. Basis rotundata, tenaculo ei thalli Holotype: Slide CR-252-2 prepared from a

TABLE 2 Costa Rican Trichomycetes (Harpellales and Amoebidiales) and their insect larval hosts1

Harpellales Harpella tica sp . nov. Simuliidae Genistellospora guanacastensis sp. nov. Simuliidae Genistellospora homothallica Simuliidae Genistel/ospora nubita sp. nov. Simuliidae Genistellospora tepidaria sp. nov. Simuliidae Graminelloides biconica gen. et sp. nov. Simuliida e Pennella montana sp. nov. Simulíi i:lae Pennella simulii Simuliidae Simuliomyces microsporus Simuliidae Smittium annulatum sp. nov. Simuliidae Smittium culisetae Culicidae Smittium culicisoides sp. nov. Chironomidae, Simuliidae Smittium dipterorum sp. nov. Chironomidae, Simuliidae Smittiumfasciculatum Chironomidae Smittium parvum sp. nov. Chironomidae Smittium phytotelmatum Chironomidae Spartiella animae sp. nov. Baetid ae (Ephemeropte ra) Stachylina grandispora Chironomidae Stachylina nana Chironomidae Stachylina paludosa Chirononúdae Stachylina penetra lis Chirononúdae

Amoebidiales Amoebidium colluviei sp. nov. Chironomidae, Simuliidae Paramoebidium spp. Simuliidae, Ephemeroptera

1 Includ es laxa describ ed in tbis paper as well as species of Harpellales reported by Lichtwardt (1994). A key to tbe se fungal species is provided in the tex!. Chirononúdae, Culicidae and Simuliida e are families of Díptera.

Simulium sp. larva collected 16-XI-91 from Other collections: Found in many species Quebrada Cuecha (Site 6, Table 1) in of Simulium, including S. samboni Jennings, Monteverde. S. ochraceum Walker, S. metallicum group, S. •

Lichtwardt: Co st a Rican gut fungi (Trichomycetes) 1353

panamense Fairchild, S. chiriquiense Field, S. hindgut linings of three Simulium sp. larvae callidium (Dyar & Shannon), S. mexicanum collected 12-11-91 in Río Animas in northem Bellardi, a species of Subg. Psilopelmia Guanacaste Province (Site 32). One hindgut Enderlein., and other, unidentified species of contains some loose sigmoid trichospores of Simuliidae; from virtually all sites where Harpella tica. Simuliidae were found in 1984, 1986, 1988, Otber coUections: In Simulium spp. larvae and 1991. from R. Piedras (Site 30), R. Animas (32), R. Etymology: Tica, vernacular name for a Bijagua (35), and R. Naranjo (36), all collected Costa Rican. in 1991. Harpella tica was the only species of that Etymology: After Guanacaste Province, genus found in Costa Rica. With very few where the fungus wasfound. exceptions, all dissected simuliid larvae were Prior to this description and those that fol­ infected. The most obvious identifying charac­ low, Genistellospora was a monotypic genus ter of H. tica is the sigmoid shape of the tri­ (based on G. homothallica Lichtw.). Though chospores (Figs. 2-6). In a few of the many zygospores were not found in G. guanacasten­ hundreds of simuliid larvae examined, the tri­ sis so as to confirm the generic designation, chospores were curved or almost coiled, which this is clearly a species of Genistellospora, is the typical shape in theother three species of which is characterized by a branched thallus Harpella. Harpella melusinae Léger & consisting of a main axis and a secreted hold­ Duboscq, has been found in many parts of the fast structure (Figs. 7, 8), and producing ovoid world (throughout Europe, North Atnerica, trichospores with multiple fine basal Japan, Australasia), has trichospores that are appendages (Fig. 10). Trichospores of G. considerably larger (-150 x 6-10 Ilm) than guanacastense are much longer than those of those of H. tica, and can be identified most the type species, which measure (24-)30(-41) easily by the tapered holdfast appar¡ttus x (8-)10(-13)J.lID. The appendages oí (ReichIe and Lichtwardt, 1972). Harpella lep­ Genistellospora trichospores before release can tosa Lichtw. & S.T. Moss, currentIy known be seen within the generative cell as straight, only from a few streams in northem Montana, parallel lines (Fig. 9) lying adjacent to !he cell U.S.A., has a base that is slightly tapered aboye wall (and outside the plasmalemma) (Moss and the small holdfast, and has trichospores that Lichtwardt, 1976). However, upon release of measure 110-150 x 4.5 Ilm. Harpella meridi­ the trichospore, the ends of the appendages of analis Lichtw. & Arenas was recentIy G. guanacastensis arecoiled (Fig. 10). described froro southem Chile (Lichtwardt and Arenas, 1996) as having a rounded base and tri­ Genistellosporanubila Lichtw., sp. nov. chospores measurlng80-100 x 4-8Ilm. (Figs. 11-13)

Genistellosporaguanacastensis Lichtw., sp. nov. Thallus 400-1200Ilm, tenaculo parco secret. (Figs. 7-1O) Trichosporae ovoideae, 38-41 x 10-14 J.lm, inoc­ ulator cellulae genital affix, liberate appendicuHs Thalli stirps praecipua plerumque 150-250 4(-6) praeditae. Zygosporae ignotae. In lar­ IlID longa, origo ramorum et lateralium et ter­ varum Simuliidárum proctodaeo. Typus: FH minaliutn. Trichosporae elongato-ovales, 50- (CR-198-6). 61 x (8-)10(-13) Ilm, appendiculas 4(-6) partim Thallus 400-1200Ilm with a small secreted conspiratas gignentes. Zygosporae ignotae. In holdfast Trichospores ovoid, 38-41 x 10-141lm, larvarum Simuliidatumpróctodaeo. Typus: FH attached eccentrically to generative cell,' on (CR-247-4). release having 4(-6) appendages. Zygospores Thallus with a main axis usually 150-250 unknown. In hindgut of Simuliidae larvae. Ilm long from which lateral and sometimes Holotype: Slide CR-198-6 prepared from a terminal 'branches arise. Trichospores long­ Simulium sp. larva collected 25-VI-88 from Q. oval, 50-61 x (8-)10(-13) J.lm, bearing 4(-6) Máquina (Site 8) near the cloudforest region of partially coiled appendages. Zygospores Monteverde.' unknown. In hindgut of Simuliidae larvae. Otber collections: All from the same site Holotype: Slide CR-247-4 consisting of and date in Simulium spp. larvae. Of 54 larvae 1354 REVISTA DE BIOLOGIA TROPICAL dissected, 25 were infected with GraminelloidesLichtw., gen. nov. Genistellospora, in sorne cases too immature to verify for certainthat the fungus was G. nubila. Thalli ramis paucis praediti, basi in Etymology: L. nubes = cloud, for the cloud mucronem attenuato, mucilaginem carente. forest site. Trichosporae biconicae, sine collari, appen­ Trichospores of G. nubila have a slightly diculo singulo. Thallus totus, cellulis paucis­ lesser length to width ratio than G. homothalli­ simis basilibus exceptis, genitalis. In ca, giving them a robust appearance. The most Simuliidarum larvarumproctodaeo. obvious characteristic of this specíes is the Thalli with few branches; base tapered to a eccentric growth of trichospores from their point, without mucilage. Trichospores biconical, generative cells (Figs. 11, 12). collarless, with a single appendage. Entire thal­ lus may become reproductive except for the very Genistellospora tepidaria Lichtw., sp. nov. basal cells. In hindgut of Simuliidae larvae. (Figs. 14-18) Basionym: Graminelloides biconica Lichtw. Thalli basis tenaculo prorninence secret prae­ Etymology: Resembles the harpellid genus dia. Thallus ramose paucos vel multos gignens. Graminella, which also produces long series of Trichosporae ovoideae, 21-31 x 9-11 f.1m, appen­ small trichospores. diculis 6(-8) tenuibus praeditae. Zygosporae sine conjugationibus productae, 58-70 x 10-15 f.1m. Graminelloidesbiconica Lichtw., sp. nov. Zygosporophorae 29-30 x 8-11 !lm. In larvarum (Figs. 19-23) Simuliidarum proctodaeo. Typus: FH (CR-221- 1). Thalli maturi usque ad 350 f.1m longi, 5-8 Base of thallus with a prominent secreted f.1m diametro, ramis sparsis, immaturi holdfast. Thallus with few to numerous branch­ fusiformes. Basis attenuata, sine mucilagine. es. Trichospores ovoid, 21-31 x 9-11 !lm, with Thalli saepe fasciculatae, nonnumquam pIures 6(-8) fine appendages. Zygospores produced quam 24 in circulo. Trichosporae biconicae, without conjugations, 58-70 x 10-15 f.1m. 10-16 x 4-5 f.1m, sine collari, appendiculo sin­ Zygosporophores 29-30 x 8-11 f.1m. In hindgut gulo brevi. Zygosporae ignotae. In of Simuliidae larvae. Simuliidarum larvarum proctodaeo. Typus: FH Holotype: Slide CR-221-1 prepared from (CR-65-1). three Simulium sp. hindguts, one with a large Mature thalli up to 350 !lm long by 5-8 f.1m thallus of Genistellospora tepidaria with numer­ diameter, with sparse branching. Immature ous trichospores, collected 23-X-91 from Río thalli fusiform. Base tapered, without Sábalo, La Selva Biological Station (Site 3). mucilage. Thalli often fascicled, sometimes Paratype: Slide CR-5-1O with pieces of many consisting of more than 24 thalli in a cluster. Simulium sp. hindguts, one with a thallus with Trichospores biconical, 10-16 x 4-5 !lm, with­ two zygospores and a few trichospores, collected out a collar, and with one short appendage. 21-IX-84from samesite as the holotype. Zygospores unknown. In hindgut of Otber coUections: Genistellospora tepidaria Simuliidae larvae. was found frequently in Simuliidae larvae from Holotype: Slide CR-65-1 prepared from the Sites 1, 2, 3, 4 in 1984, 1986, 1988 and 1991 (not hindgut of an unidentified Simuliidae larva col­ necessarily all sites in the same year). The hosts lected 25-XI-84 in an unnamed stream W of included, in addition to unidentified species, S. Portalón (3.3 km E on the road to Portalón samboni, S. ochraceum, S. mexicanum, and from junction of main road) and SE of Quepos Subg. Psilopelmia sp. in Puntarenas Province (general Site 25). The specific epithet (L. tepidus = warm) Otber collections: In Simulium callidium refers to the relatively warm streamtemperatures and an unidentified species of Simuliidae (24-25.8 C) where the blackfly larvae were liv­ (same as holotype), from same site and date. ing. Genistellospora tepidaria is most similar to Seven of 26 simuliid larvae were infected G. nubila, but has smaller trichospores. with the new fungus. The features that distín­ Trichospores were sometimes attached eccentri­ guish this new monotypic genus from two other cally, as they usuallyare in G. nubila. harpellid genera that produce trichospores with Lichtwardt: Costa Rican gut fungi (Trichornycetes) 1355 no collar and a single appendage indude: (i) Branch, where the fungi were immature) in the the tapered base of the thallus (Figs. 19, 20), Wilson Botanical Garden in 1991 (Site 27), in (ii) the biconical shape of the trichospore (Figs. S. ochraceum, S. callidium, S. chiriquiense, S. 21-23), (iii) a thallus that becomes almost metallicum group, and unidentified blackfIy entirely reproductive (Fig. 22), and (iv) the species. dipteran host. The two other genera with col­ Pennella montana (L. montanus = moun­ larless trichospores and a single appendage live tain) appears to be a fungal species that is limit­ in Baetidae (Ephemeroptera): species of ed to higher altitudes. It was not found in low­ Graminella ex Manier have bulbous thallic land streams, including those in Guanacaste bases, ovoid trichospores, and produce vegeta­ Province. Its larger trichospores and tive propagules; and the currently monotypic zygospores distinguish it from the other six genus Spartiella ex Manier (but see new described species of Pennella ex Manier. An species below) produces obpyriform tri­ unusual feature of P. montana is the manner in chospores and the thallus has a swollen or lob­ which zygospores formo A special cell from an ulate base. The fascicled nature of adjacent branch, the conjugation cell (Fig. 29), Graminelloides biconica thalli is unusual (Figs. produces an extension that brings about a con­ 19, 20), because the origin of each thallus is jugation with the branch that will become the presumed to be from a separate trichospore that zygosporophore from which the zygospore is ingested by the host. develops. The conjugation cell subsequently breaks loose from the branch that produced it, Pennella montana Lichtw., sp. nov. but remains attached to the zygosporophore (Figs. 24-30) (Fig. 30).

Thallorum maturorum bases semel vel Smittium annulatum Lichtw., sp. nov. iterum dichotome bifurcatae, mucilaginem (Figs. 31-33) secretentes. Trichosporae ovoideae, (39-)60(- 72) x 8-10 J.lm, appendiculis pertenues praedi­ Thalli parvi compactique, structura promi­ tae. Zygosporae 91-100 x 1 8 -20 J.lm; nence . basali cellulis sex in annulo ordinatis zygosporophoris 38-46 x 13-16 J.lm. In lar­ constante orti. Trichosporae elongato-ovales, varum Simuliidarum proctodaeo. Typus: FH 18-22(-33) x 4-6(-8) J.lm, collari 3-6 J.lmlongo . (CR-30-1). Cellulae genitales usque ad 8 vel pIures in Bases of mature thalli dichotomously ramo fertili uno gestae. Zygosporae ignotae. bifurcate one ormore times, producing a In proctodaeo larvarum Simuliidarum. Typus: mucilaginous secretion. Trichospores long­ FH (CR-143-5). ovoid, (39-)60(- 72) x 8-10 J.lm, with multiple Thalli small and compact, arising from a very fine appendages. Zygospores 91-100 x prominent basal structure consisting of about 18-20 J.lm; zygosporQphores 38-46 x 13-16 six cells arranged in a ringo Trichospores elon­ J.lm. In.hindgut of Simuliidae larvae. gafe-oval, 18-22(-33) x 4-6(-8) J.lm, collar 3-6 Holotype: Slide CR-30-1 prepared from a J.lm long. Up 10 8 or more generative cells per Simulium ochraceum larva collected 16-X-84 fertile branch. Zygospores unknown. In from a very smaU headwater stream crossing hindgut of larval Simuliidae. Sendero Bosque Nuboso in the Monteverde Holotype: Slide CR-143-5 prepared from a Cloud ForestReserve (Site 9). Consists of sev­ Simulium sp. larva (possibly S. ochraceum eral mature thalli with trichospores, one with Walker) collected 27-V-88 in Q. Esquina (Site zygospores. The slide also contains a per­ 2), at the La Selva Biological Station. itrophic membrane with Harpella tica and Otber collections: In Simulium samboni sorneloose H. . tica trichospores. Jennings larvae collected at Site 1 in 1984 and Otber collections: In many species of Sites 1 and 2 in 1988. Simuliidae larvae in the Monteverde area in Cultures: CR- 143-8 and CR-143-10 (the 1984, 1986, 1988, 1991 (Sites 6, 7, 8, 9), latter is no longer living) isolated 27-V-88 from streams along the Cordillera de Talamanca in Simuliidae larvae (possibly S. ochraceum) 1984 (Sites 18, 19, 20, 21), and Snake Creek fromthe same site as the holotype. (possibly also Lutite Springand R. Jaba East The ring-like structure at the base of the 1356 REVISTA DE BIOLOGIA TROPICAL thallus (Fig. 31) (to which the specific epithet simuliid larvae). The name of this species refers), as well as the dimensions of the tri­ anudes to its simílarity withS. culicis Manier, chospores, make this species easily identifiable. with which it can be confused. Smittium A study of isozyme patterns in cultured culicis, a common and widely distributed Trichomycetes (Grigg, 1994; Grigg and harpellid, is most often found growing in mos­ Lichtwardt, 1996) demonstrated that S. annula­ quito larvae (Culicidae), but has a broad host tum ís isozymically the most distinct of the range that occasionally ineludes other dipteran Costa Rican species of Smittium (Fig. 79). It families, including Chironomidae and was relatively common in Sites 1 and 2, but, Simuliidae. The trichospores of S. culicis are with one exception in 1984, was not found in often formed on arching terminal branchlets, other simuliid larvae despite a large number of and the branching within the thallus is more dissectíons. Nor was S. annulatum found in divergent than in S. culicisoides. The tri­ other Costa Rican streams used in this study. chospores of both species are very similar in their shape and dimensions, with those of S. Smittium culicisoides Lichtw., sp. nov. culicisoides having, on the average, a slightly (Figs. 34-38) lesser length to width ratio. That these cryptic species are distinct Ís supported by differences Thalli maturi saepe 200-500 !lm longi. in their isozymic patterns (Fig. 79). Bases attenuatae, nonnumquam prominentiis ex muro minutis ornatae, in hospitibus forte infec­ Smittium dipterorumLichtw ., sp. nov. tis aliquando fasciculatae. Trichosporae (Figs. 39-45) ovales, 20-25(-28) x 6-10 !lm, collari 5-10!lm . Rami fertiles cellulas genitales 1-4 gerentes. Thallorum tenaculum simplex secretum, Zygosporae ignotae. In proctodaeo larvarum rami crebre verticellati,omnes fertiles usque ad Chironomidarum et Simuliídarum. Typus: FH 8 vel pIures cellulas genitales gerentes. (CR-llO- 19). Trichosporae longe ellipsoidales, subcylindri­ Mature thalli often 200-500 !lm long. cae, (10-)12-18(-26) x 2-3(-4) ¡..tm, appendiculo Bases tapered, occasionally with minute pro­ pertenui, collari 1-3!lm longo. Zygosporae 51- jections from the wall. Bases may occur in fas­ 70 x 9-12 !lm. In proctodaeo larvarum cicles in well infected hosts. Trichospores Simuliidarum et Chironomidarum. Typus: FH oval, 20-25(-28) x 6-10 !lm, collar 5-10 !lm. (CR-260-2). Fertile branches with 1-4 generative cells. Thalli with a simple secreted holdfast, Zygospores unknown. In hindgut of larval branches oftenverticillate with up to 8 or more Chironomidae and Símuliidae. generative cells per fertile branch. Holotype: Slide CR-IlO- 19 prepared from Trichospores long-eUipsoidal, almost cylindri­ a Cardiocladius sp. (Chironomidae) larva col­ cal, (10-)12-18(-26) x 2-3(-4) ¡..tm, with a very lected 16-VIl-86 from a polluted stretch of Río fine appendage; collar 1-3 !lm long. Guacimal (Site 7) in Monteverde. The slide Zygospores 51-70 x 9-12 !lm. In hindgut of also contains a small amount of S. dipterorum larval Simuliidae and Chironomidae. with a zygospore. Holotype: Slide CR-260-2 (with tri­ Other coUections: All from Site 7, in 1984, chospores) prepared from a Simulium sp. 1986, 1988, and 1991, predominantIy found in (Simuliidae) larva collected ll-XI-91 from an larvae of Cricotopus sp., but also in unpolluted stretch of Río Guacimal (Síte 7). Chironomus sp. and other Chironomidae, and SUde also contains Pennella sp. and Simulium sp. (S�muliidae). Genistellospora sp. Paratype: Slide CR-17-3 Culture: CR-253-12 isolated from an unde­ (includes zygospores) prepared from an termined species of Chironomidae collected Orthocladius (Euorthocladius) (Chironomidae) 17-XI-91. larva collected 9-X-84 from Río Chorreras Etymology: Resembles Smittium culicis. where it crosses Rt. 9 NW of Heredia (Site 13). Smittium culicisoides was common in many Types deposited at Farlow Herbarium (FH). larvae at the type site, often in association with O ther collections: In many streams, S. dipterorum (see below), and other harpellids including Sites 1, 2, 7, 8, 9, in various (Harpe lla, Genistellospora, or Pennella in Simuliidae (Simulium samboni, S. callidium, Lichtwardt: Costa Rican gut fungi (Trichomycetes) 1357 and unidentified species), and Chironomidae (FH). (Cardiocladius sp., Cricotopus sp., Other collections: Culture CR-254-10 Orthocladius sp., and unidentified species), in (Fig. 49) isolated 18-XI-91 from a Cricotopus 1984, 1986, 1988, and 199L sp. larva from the same site as the Holotype. Cultures: CR- 141-17, isolated 26-V-88 Possibly also in a Cricotopus sp. larva coUeét­ from Simulium sp., Site 1; CR-143-2, 27-V-88 ed 21-V -88 from Quebrada Sábalo (Site 3) (no longer living) from Simuliüm sp., Site 2; (Fig. 46). CR-253-14, 18-XI-91 from Simulium sp., Site Cultures: CR-1 84-32 isolated 18-Vl-88 7; CR-254-13, 18-XI-91 fromChironomus sp., from a Cricotopus sp. larva' collected in Río Site 7. Guacimal (Site 7) (culture no longer living); Simulium dipterorum was found in more CR-254- 1O isolated 18-XI-91 from a Costa Rican streams and a greater variety of Cricotopus sp. larva at Site 7). dipteran hosts than any other species of Etymology: L. parvus = smaU, for the Smittium. Based on trichospore dimensions small trichospore size. and verticillate branching (Fig. 39); S. diptero­ Trichospores of S. parvum have the'shape rum most closely resembles S. paludis M.C. and long collar of S. culicis and S. culicisoides, Williams & Lichtw., a Tasmanian species but are smaller in size (compare Figs. 37 and found in a swamp midge. However, S. paludis 48), and is isozymicalIy rlistinct from the other has a sw�llen holdfast cell that is incurved at two Smittiums (see Fig. 79). The collection' the point of attachment; whereas in S. diptero­ from a Cricotopus larva in Q. Sábalo, La Selva rum the holdfast structure is a simple secretion Biological Station (Fig. 46), appears to· be S. and the basal cell is notswollen (Fig. 40). In parvum, and this may be the only specimen the Monteverde collectiQns,S. dipterorum was slide prepared directly from a living host and ofien living in sorne of the same dipteran and not a culture. simuliid hosts as S. cui. ic'i$óides, as well as s,haring the simuliíd guts with Harpella, Spartiella animar! Líchtw., sp. nov. ' Genis-tellospora; or PenTÍella. (Figs: 50-53)

Smittium parvum bichtw., sp. nov. Thallis valde ramosi, quam 500!lm brev­ , (Figs. 4�-49) iores, cellula basali paulum inflata vellobulata. Trichosporae obpyrifoirnes, 13-20 x 4-7 !lm, Rami arete ordinatae, fertiles usque ad 6 vel sine colIari, appendiculum singulum longum plur�s cellulas genitales gerentes . tenue gerentes. Zygosporae ignotae . In Trichosporae ovales, 9-14 x 3.5 !lm, collari 2-6 nympharum Ephemeropteroram proctodaeo. !lm longo. Zygosporae ignotae. In larvarum Typus: FH (CR-251-1). Chironomidarum proctodaeo. Typus: FH (KU- Thalli much branched, less than 500 !lffi 204-35). long, with a slightly swollen or lobulaté' basal . Branches closely arranged, with fertile cell. Trichospores obpyriform, 13-20'x 4-7 branches bearing up to 6 or more generative !lm, collarless,bearing a single long" fine cells. Trichospores oval, 9-14 x 3.5 !lm, collar appendage. Zygospores unknown . . In hindgut , 2�6 !lm long. Zygospores unknown. In of Ephemeroptera nymphs,.. · hindgut of Chironomidae larvae. Holotype: Slide CR-25 1-1 preparedfro m a Holotype: Kodachrome transparency KU- Baetis sp. nymph (Ephemeroptera, Baetidae) 204-35, consisting of a photomicrograph taken collected 13-XI-91 in Río Animas in nortli� 14-VII-88 of culture CR-184-32 isolated 18- western Costa Rica (Site 32) near the border VI-8S from a Cricotopus sp. (Chironomidae) with Nicaragua. . larva foundliving in Río Guacimal (Site 7) 17- Other collections: Nineteen of 42 nymphs VI-8S, showing released trichospores. Isotype: of Baetis sp. contained S. animae from collec­ Kodachrome transparency KU-204-36,'a pho­ tions of 10-XI":91 and 13-XI,91 in R. Animas. tomicrograph taken on the saine date and from Also found in three of 16 mayfly nymphs of the the same culture as the HoJotype, showing tri­ same species collected 13-VI-88 in R. Piedras chospores attached to f�rtile br;mchl�ts, sorne (Site 30). released. Oepositeda{ Farlo\\' li�rbarium Etymology: Named after R. Animas, the 1358 REVISTADE BIOLOGIA TROPICAL stream where most specimens were found. amoebae were produced by A. colluviei speci­ Four attempts to culture S. animae were mens (see Discussion). One culture attempt unsuccessful. Spartiella barbata ex Manier up was not successful, though A. parasiticum to now was the only species in the genus. That Cienk. has been cultured. species has been found in Baetidae nymphs Amoebidium parasiticum has been found (Ephemeroptera) in southern France, England, in many parts of the world on the external and Wales (Lichtwardt, 1986). The genus is cuticle of small lentic crustaceans such as characterized by having obpyriform tri­ Cladocera, but also freshwater lentic insects chospores with no collar and a single such as Culícídae and bloodworms appendage, and zygospores attached perpendic­ (Líchtwardt, 1986). Sporangiospores of A. ularly and medially to the zygosporophore. No parasiticum are usualIy lunate, and measure zygospores were found in S. animae, but the 15-50 x 5-10 !lm; those of A. colluviei are trichospores differ from the type species in alIantoid and much smalIer. The fact that the being much smaller (those of S. barbata are 22- latter species was found in rapidly-flowing 27 x 7.5-10 11m). waters (Chironomus sp. larvae were mostly in sedimentary zones, however), makes this an Amoebidium colluviei Lichtw., sp. nov. unusual habitat for an Amoebidium. The only (Figs. 54-59) other species of Amoebidium described untíl now other than the type species has been A. Thalli plerumque minores quam 80 !lm recticola Chatton (1906), an apparently rare longitudine, circa 10 !lm diametro, sporan­ species descríbed from the rectal area of giosporas alIantoideis 25-32 x 6-10!lm produ­ Daphnia spp. taken from reptíle tanks at the centas. Praecipue papillae anali Paris Museum. Sporangiospores of A. recti­ Chironomidarum et Simuliidarum affixi. cola are short-cylindrical to oval, 8-12 11m Typus: FH (CR-254-22). long. Thalli usually less than 80 11m long by about 10 11m diameter, producing allantoid PROBABLY NEW BUT UNDESCRIBED sporangiospores 25-32 x 6-10 !lm. Attached SPECIES primarily to anal papillae of Chironomidae and Simuliidae larvae. Several species of Smittium were found and Holotype: Slide CR-254-22 consisting of cultured, but are not being named at this time the anal papíllae of two large Chironomus sp. because of the unavaílabilíty of sufficient num­ larvae covered with Amoebidium colluviei, bers of in vivo specimens. collected 18-XI-91 in a highly polluted stretch of Río Guacimal (Site 7). Deposited in Smittium sp. CR No. 1 Farlow Herbarium (FH). (Hg. 60) Other collections: Common on anal papil­ lae of Chironomus sp. and Simulium spp. in This Smittium sp. was found in Tanytarsus the same collection site as the Holotype. sp. larvae in a rock pool about 0.5 m aboye the Etymology: L. colluvieis = polluted, for stream water in R. Piedras (Síte 30), and possibly the condition of the stream site. also in the same host in the river channel. The Amoebidiales, as mentioned in the rock pool was subject to periodic flooding. Introduction, are not thought to be Trichospores of the gut fungus are cylindrical, Trichomycetes, but are often found living in 19-21 (-30) x 2.3 !lm,wíth a collarabout 2-3 !lm (Paramoebidium spp.) or on (Amoebidium long. The trichospores are similar to those of S. spp.) sorne of the same kinds of hosts that are phytotelmatum Lichtw., a species found at La inhabited by trichomycete gut fungi. The Selva most commonly in bloodworrns inhabiting external thalli of A. colluviei may have been water at the base of bromeliad leaves present but not detected in other years when (Lichtwardt, 1994), but the branching pattern is the author colIected in the same polIuted sec­ different. An axenic culture, CR-239-12, was tion of Río Guacimal. It pro vides a fuzzy made from one of the Tanytarsus larvae collect­ covering to anal papillae of infested chirono­ ed 9-XI-91. A study of isozyme patterns in cul­ mid and, to a lesser extent, simuliids. No tured Harpellales (Grigg and Lichtwardt, 1996) Lichtwardt: Costa Rican gut fungi (Trichornycetes) 1359 showed thatSmittium sp. CR No. 1 was isozymi­ atively cornmon and widespread in a variety of cally distinctfrom S. phytotelmatum isolates and Simulium and Prosimulium species in the U.S.A., otherharpellid species (Fig. 79). It appears to be especially in theRocky Mountains, and has been a new, undescribedgut fungus. Host larvaewere reported from southern England (Lichtwardt, more often infected with a species of Stachylina 1986). More recently it was found in southero than with this Smittium. Chile (Lichtwardt and Arenas, 1996) and in Argentina (López Lastra, unpublished). Smittium sp. CR No. 2 Collections of G. homothallica in Costa Rica (Fig. 61) were in higher altitudes: the Monteverde region (Sites 6, 7, 8, 9) and the Cordillera de Talamanca Simulium sp. larvae collected 21-XI-91 from (Sites 18, 21). The Costa Rican specimens had Q. Máquina (Site 8) were infected with Smittium trichospores (measuring 29-36 x 10-11 flm) that sp. CR No. 2, and an isolate, culture CR-259-4, were sometimes eccentrically attached andhad a was obtained (Fig. 61). The culture is isozymi­ somewhat lesser length to width ratio than nor­ cally distinct from other Costa Rican Smittiums mally seen in other collections, and the (Fig. 79) as well as Smittium spp. from othergeo­ zygospores (measuring 83-90 x 12-17 flm) were graphic areas (Grigg and Lichtwardt 1996). a bit narrower than thosefound in most othercol­ Morphologically, the oval trichospores, measur­ lections. However, the Costa Rican specimens ing 19-27 x 4-8 flm with a collar about 2-4 flm generally fit the description of G. homothallica, long, have sorne resemblance to S. culicisoides which has the following published measure­ and S. culicis, but are somewhat smaller, have a ments: trichospores (24-)30(-41) x (8-) 1O( -13) greater length to width ratio, and the collar is flm; zygospores (77-)1 00(- 113) x (15-)20(-22) generally shorter. In vivo sporulating specimens flm. are requiredbefore it is described. Paramoebidium spp. Smittium sp. CR No. 3 (Fig. 69) (Figs. 62, 63) Though not believed to be phylogenetically The third undescribed-and probably new­ related to fungi, as explained in the introduction, species of Smittium, thoughit carnefrom a lentic, species of Paramoebidium may be common in rather than a riparian, bloodworm, is illustrated Ephemeroptera, Plecoptera, and Simuliidae, and here as culture CR-21l-1. It was living in a may coinhabit guts with Harpellales. Only six Chironomus sp. larva in a swamp near Sendero valid species of this unculturable genus have ExperimentalSur at La Selva. Lichtwardt(1 994) been named. None of the Costa Rican species originallysuggested that it might be similar to S. appeared to be among the currently described phytotelmatum, but this does not now appear to species, and none is described in this paper be the case, either morphologically or isozymi­ because of a lack of suitable descriptive charac­ cally (Fig. 79). Figures 62 and 63 illustrate sorne terso Nonetheless, Paramoebidium spp, were aspects of its morphology. Trichospores are found in most collections of mayflies (Baetidae) elongate elliptical, 15-18 x 3-4 flm, with a very in many sites representing different life zones, short collar (-1 flm long). The similarity in and somewhat less cornmonly in many popula­ isozyme pattero with culture CR-253-12, S. culi­ tions of Simuliidae larvae. Thalli of cisoides, cannot be explained, because the two Paramoebidium may be quite !arge and numer­ aremorphologically distinct. ous in sorne hosts. They are more or !ess cylin­ drica!, straight or curved or bent (Fig. 69), non­ SPECIES WITH WIDERGEOGRAPHICDIS­ septate, and mature thalli occasionally may be TRIBUTIONS seen releasing large numbers of amoeboid cells when a larva is dissected or is in the process of Genistellospora homothallicaLichtw. molting. The various stages of this interesting (Figs. 64-68) protist have been described and illustrated (e.g. Lichtwardt 1986; Lichtwardtand Aenasr 1996). Priorto this publication, G. homothallica was the only described species of the genus. It is rel- 1360 REVISTA DE BIOLOGIA TROPICAL

Pennella simulii M.C. Williams & Lichtw. in Costa Rica, however. It was found on the (Fig. 70) peritrophic membrane of Chironomus sp. lar­ vae in polluted stretches of R. Guacimal (Site 7) On two occasions at La Selva (Sites 1 and in 1986 and 1991, and bloodworms in a 2), in 1984 and 1991, within Simulium barely flowing cree k in Santa Rosa National 33). ochraceum and possibly another blackfly Park (Site Stachylina grandispora, species, a Pennella was. found with tri­ among other characters, produces large tri­ chospores resembling the size and shape of P. chospores (40-72 x 6-10 J.lm) that usually simulii. No zygospores were found on either have a short but visible collar. The Costa occasion for substantiation of the identifica­ Rican specimens tended to have smaller tri­ tion. Pennella simulii has been reported from chospores than usual and had a slightly the Rocky Mountains of Colorado and greater length to width ratio, but generally Wyoming, U.S.A. (Lichtwardt, 1986), but fell within the size range for S. grandispora. probably has a much broader range of distrib­ The possibility that those specimens represent ution in North America. Measurements of P. a new species cannot be exc1uded. simuUi are: trichospores (30-) 33 (-41) x (6.5-) 8 (-1 0.5) ¡.tm; zygospores (84-) 90 (-96) Stachylina nana Lichtw. x (19-) 22 (-24) ¡.tm. (Figs. 76, 77)

Simuliomyces microsporus Lichtw. First described from France (Lichtwardt (Figs. 71, 72) 1984), Stachylina nana has been reported from Japan (Lichtwardt et al. 1987), New Simuliomyces microsporus is a wide­ Zealand (Williams and Lichtwardt 1990) , spread Harpellales that is sometimes confused and now from Costa Rica. Trichospore sizes with Smittium when zygospores are not pre­ are somewhat variable, ranging from 25-40 x sent. Trichospores of Smittium spp. have a 7-11 J.lm, but the thallus is distinctively collar and one appendage. The 2-4 cymbiform to fusiform and produces 2-4(-8) trichospores. In Costa Rica it was found on appendages of S. microsporus often are not 7) c1early visible on released trichospores, and several occasions in Río Guacimal (Site the trichospores are collarless. Simuliomyces predominantly in Cricotopus sp. but also in microsporus is easily recognized, however, Chironomus sp. larvae, and what may be the Stachylina because of its common attachment to thalli of same species of was seen once in a Orthocladius (Euorthocladius) other harpellids in simuliid hindguts, such as larva of sp. 17). Pennella or Genistellospora, but most often it collected in Río Chorreras (Site is seen attached to thalli of Paramoebidium spp. Simuliomyces microsporus was found in Stachylína penetralis Lichtwardt the following sites: 7, 9, 12, and 18. The (Fig. 78) published measurements of this specíes are: trichospores 20-30 x 4-6 J.lm; zygospores 34- Stachylina penetralis is one of several 45 x 7-9 J.lm. Stachylinas whose bases penetrate through the loose peritrophic membrane that lines the Stachylina grandispora Lichtw. midgut. In this species the thallus has a bul­ (Figs. 73-75) bous base and produces 2-12 long-ellipsoidal trichospores that range in size from 30-50 x Stachylina grandispora is perhaps the 8-12 ¡.tm. Only one specimen was found. It Cricotopus most widespread of all species of the genus, was in a sp. larva on the divide of having been found in at least six genera of the Cordillera de Talamanca at the initiation midge larvae, but especially bloodworms of of the West Branch of Q. Macho Mora (Site 20). the genus Chironomus, in Europe, U.S.A. The species has been found on several (including Hawaii), Japan, Australia, and continents. New Zealand. The species was not common Lichtwardt: Costa Rican gut fungi (Trichomycetes) 1361

Figs. 2-6. Harpella tica. 2. Two sporulatingthalli within the transparent peritrophic membrane of a Simuliidae larva. 3-5. Segments of a single thallus attached to a peritrophic membraneby a secreted holdfast (single arrow), the teoninal trichospore havingjust released and with sorne of the 2-4 very fine basal appendages barely visible (double arrows). 6. Typical sigmoid trichospore with part of twoof the basal appendages in focus (arrows), Fig. 2 bar = 80 ¡lm ; Fig. 3 bar = .40 j,1m(same magni- fication forother Figures). " 1362 REVISTA DE BIOLOGlA TROPICAL

Figs. 7-10. Genistellospora guanacastensis. 7. Sporulating thallus removedfrom the hindgut cuticle of a Simuliidae larva. 8. Base of a thallus attached to the host cuticle by means of a secreted holdfast (arrow). 9. Sporulating branchlet showing appendages within the generative cell (arrow). 10. Released trichospores with multiple coiled basal appendages. Fig. 7 bar =

80 flm; Fig. 8 bar = 40 flm (sarne magnification forother Figures). Lichtwardt: Costa Rican gut fungí (Trichornycetes) 1363

Figs. 11-13. Genistellospora nubila. 11. Entire sporulating thallus. 12. Trichospores,one released and showing several shad­ owy appendages attached to its base (arrow). 13. Initiation of trichospotes at the tips of branchlike extensions. Fig. 11 bar :;: 40 �m (same rnagnificationfor Fig. 13); Fig. 12 bar :;:40 ¡¡m. 1364 REVISTA DE BIOLOGIA TROPICAL

Figs. 14-18. Genistellospora tepidaria. 14. Sporulating thallus attaehed to a pieee of hindgut eutiele. 15. Trichospores. 16.

Released trichospore with six barely visible appendages. 17, 18. Zygospores formed without conjugation. Fig. 14 bar = 80

J.1m;Fig. 16 bar = 40 J.1m(same magnificationfor other Figures). Lichtwardt: Costa Ricangut fungi (Trichomycetes) 1365

Figs. 19-23. Graminelloides biconica. 19. Fascicled (clustered) arrangement of immature thalliremoved fromthe hindgut of a Simuliidae larva. 20. Tapered bases of sporulating thalli attached to a piece of hindgut lining. 21, 22. Trichospores pro­ duced by a sparsely branched thallus; note almost complete conversion of thallus to trichosporeproduction. 23. Released tri­ chospore with a short single appendage. Fig. 19 bar = 40 �m (same magnification for Fig. 22); Fig. 21 bar= 40 ¡.1m (same magnificationfor Figs. 20, 23). 1366 REVISTA DE BIOLOGIA TROPICAL

Figs. 24-30. Pennella montana. 24. Two attached trichospores. 25. Released trichospore showing one of the multiple appendages in focus (arrow). 26-28. Bases of mature thalli with mucilaginous secretion and with different degrees of bifur­ cation. 29. Conjugation oftwo branches (long arrow), a stage that initiates zygospore formation; within ¡he receptive cell that will become the zygosporophore are seen two nuclei (short arrows) prior lo their fusiono 30. Zygospore attached 10 the zygosporophore, with Ihe attached conjugation cell (arrow) now broken loose from the branch Ihat produced il. Fig. 25 bar = 40 ¡.1m (applies to all Figures). Lichtwardt: Costa Rícan gut Cungí (Trichomycetes) 1367

Figs. 31-33. Smittium annu[atum. 31. Small, compact thallus attached to the hindgut cuticle oC a Simuliidae larva by a rin� oC enlarged cells. 32, 33. Attached and loose trichospores from a pure culture (CR-143-8). Scale bar = 40 Ilffi (applies to all Figures). 1368 REVISTA DE BIOLOGIA TROPICAL

Figs. 34·38. Smittium culicisoides. 34. Part of the dissected hindgut of a Chironornidae larva packed with fungal thalli. 35, 36. Sporulating branchlets. 37. Detached trichospores from a pure culture {CR·253"12). 38. An immature thallus showing the

tapered basal parto Fig. 34 bar = 80 ¡.¡m; Fig. 36 bar= 40 ¡.¡m (same magnification for other Figures). Lichtwardt: Costa Rican gut fungi (Trichomycetes) 1369

Figs. 39�45. Smittium dipterorum. 39, 40. Sporulating thalli showing verticillate branching and a simple holdfast (arrow). 41, .Detached trichospores from a pure culture(CR- 253-14) isolatedfrom a Simuliidae larva. 42. Culture CR-254-13 isolated rrom a (::hironomidae ¡aÍva. 43. Maturing zygospore. 44-45. Attached and detached mature zygosporéS from lactophenol éoUQn-blue mounts, oné sbowing a collar and one short appendage (arrow). Fig. 41 scale bar = 40 ¡¡m (applies to all Figures). 1370 REVISTADE BIOLOGIA TROPICAL

Figs. 46-49. Smittium parvum. 46. Part of a thallus with developing trichospores removed from the hindgut of a Chironomidae larva. 47. Attached and detached trichospores reproduced from the Isotype Kodachrome transparency (culture CR-1 84-32). 48. Trichospores from the Holotype transparency (culture CR-184-32). 49. Trichospores from culture CR-254-

10. Fig. 46 bar =: 40 Ilm(same magníficatíonfor all Figures). Lichtwardt: Costa Rican gut fungi (Trichornycetes) 1371

Figs. 5()"53. Sp artiella animae. 50. High1y branched thallus attached to hindgut cuticle of a rnayt1y nyrnph. 51. Holdfast region of sarnetha llus. 52, 53. Attached and loose trichospores. Fig. 50 bar= 80 I!m ; Fig. 53 bar= 40 I!rn (sarnernagnifica­ tion Cor other Figures). 1372 REVISTA DE BIOLOGIA TROPICAL

Figs. 54-59. Amoebidium colluviei. Anal tubule of a bloodworm (Chironomus sp.) completely covered with thalli of A. col­ luviei in different developmental stages. 55. Attached mature thalli, the one on the right containing sporangiospores. 56, 57. Detached thalli showing small holdfast (arrows), one with sporangiospores. 58, 59. Released allantoid sporangiospores. Fig. 54 bar = 80 ¡,¡m ;Fig. 55 bar= 40 .¡,¡m (same magnificationfor other Figures). Lichtwardt: Costa Ricangut fungi (Trichomycetes) 1373

Figs. 60-63. Unnamed, possib1y new Smittium spp. in axenicculture. 60. Smittium sp. CR No. 1 (culture CR-239-12). 61. Smittium sp. CR No. 2 (cultureCR-259-4) . 62, 63. Smittium sp. CR No. 3 (cultureCR- 211-l). Bar= 40 ¡.¡mfor aH Figures. 1374 REVISTA DE BIOLOGIA TROPICAL

Figs. 64-68. Genistellospora homothallica fromhindgut of Simuliidae larvae. 64. Base of a sporulating thallus with a pronú­ nent secreted holdfast. 65. Developing trichospores. 66. Rare 2-celled thallus with irnrnature trichospores. 67. Trichospores

and 5 biconical zygospores. 68. Two zygospores which, in trus genus, form without prior conjugation of branches. Fig. 64 =

40 11m ; same magnification for other Figures except Fig. 67, where bar = 40 11m . Lichtwardt: Costa Ricangut fungi (Trichomycetes) 1375

Fig. 69. Paramoebidium sp. growing within the hindgut of a mayfly nymph. Fig. 70. Part of a Pennella simulii thallus with recently released trichospores with multiple basal appendages. Figs. 71, 72. Trichospores and zygospores of Simuliomyces microsporus. Figs. 73-75. Thalli and loose trichospores of Stachylina grandispora witbin the peritrophic membrane of a bloodworm (Chironomus sp.). Fig. 69 bar== 40 ¡.lm .; Fig. 73 bar == 80 ¡.lm; Fig. 70 bar == 40 ¡.lm (same magnification for other Figures). 1 3 76 REVISTA DE BIOLOGIA TROPICAL

Fig. 76. 77. Stachylina nana within the peritrophic membrane of a Chironomidae larva. 76. Three sporuJating thalli. 77. Peritrophic membrane with a Jarge number of individual sporulating thalli; each thalJus deveJoped from one ingested tri­ chospore. Fig. 78. SporuJating thallus of Stachy/ina penetra lis whose slightly bulbous base has penetrated the peritrophic membrane. Fig. n bar = 80 ¡.lm ; Fig. 78 bar = 40 ¡.lm (applies also to Fig. 76). Lichtwardt: Costa Rican gut fungi (Trichomycetes) 1377

0.00 0.25 0.50 0.75 1.00

...--- -;::::==:c======\N'{G 51·1 1 S. CLlicis ml�8 S. snJatun r-----{==== m21 1·1 Snittiunsp. #3 m253- 12 S. CLlicisoides

1--______m259-4 Snittiunsp. #2 r------m23S-12 Snittiunsp. #1 m254-1O S. parvun m253-14 S. dpteroom m254-13 S. dpteroom m219-1 S. ¡j1ytotelrratun m219-3 S. ¡j1ytotelrratun

Fig. 79. Phenogram derived from an analysis of isozyme pattems produced by 10 cultured isolates of Smittium from Costa Rica (CR) and one from Wyoming, U.S.A. (WYO). Dipteran hosts of the CR species areprovided in Table 1 and in the texto SmiUium culicisoides (CR·253·12), from a Chironomidae larva, produces trichospores that areessentially identical to S. culi· cis, represented here by isolate WYO-51-lt from a Culicidae larva, but they can be seen to be isozymically distinct. The similarity of isozyme pattems between S. 'culicisoides (CR-253-12) and Smittium sp. #3 (CR-211-l) is not explainable, because the two species are morphologically quite different. Scale at top represents a numerical measure of similarity of isozyme pattems. Analysis based on isozyme data obtained by Grigg (1994).

KEY TO TRICHOMYCETES ASSOCIATEDWITH COSTA RICAN AQUATIC INSECTS

1- Small cylindrical thalli « 100 mm long) attached externally to the anal papillae

of Chironomidae or Simuliidae larvae ...... Amoebidium colluviei (Amoebidiales) 1'. Thalli attached internally to hindgut cutic1e or peritrophic membrane ...... , ...2 2(1 '). Thalliunbranched attached to peritrophic membrane of Diptera larvae ...., ...... 3 2' (F). Thalli branched or unbranched, attached to hindgut cuticle of Diptera or

...... Ephemeroptera ...... 7 3(2). In Simuliidae larvae; trichospores cylindrical, sinuate or more rarely curved, with 2-4 appendages ...... : ...... Harpella tica

...... 3'(2). In Chironomidae larvae, trichospores ellipsoidal, straight, with 1 appendage ......

...... 4 4(3'). Base of thallus bulbous, protruding through the peritrophic

...... membrane ...... Stachylina penetra lis 4'(3 '). Base of thallus with a small holdfast, not penetrating the

...... peritrophic membrane ...... , ...... 5 5(4'). Trichospores without a collar,25-40 x 7-11 Ilm ...... Stachylina nana 5'(4').Trichospores with a very short collar...... :: ...... 6 6(5'). Trichospores 40-70 x 6-10 Ilm ...... st achylina grandispora

6(5'). Trichospores 31-40 x 6-8 Ilm...... stachylina paludosa

...... 7(2'). In Ephemeroptera nymphs ...... Sp artiella animae 7'(2').1n Diptera larvae ...... 8 8(7'). Thalliunbranched, nonseptate, cylindrical or sausage-shaped, straight throughout or curved, with a distinct basal holdfast; reproducing

...... by release of amoeboid cells ...... Paramoebidium spp. (Amoebidiales) 8' (7'). Thallibranched, septate, attached by a secreted haldfast or by a basal mucilaginous secretion; producing trichospores laterally on fertile

...... branches, and sometimes zygospores ...... 9

(Continued... ) 1378 REVISTA DE BIOLOGIA TROPICAL

9(8'). Trichospores biconical, without a collar,wit h 1 basal appendage; in Simuliidae larvae ...... Graminelloides biconica 9'(8').Trichospores not biconical; either with a collarand 1 basal appendage or without a collar and 2-6 basal appendages ...... 10 10(9'). Trichospores without a collar, with more than one basal appendage; in Simuliidae larvae...... 11 10'(9'). Trichospores with a collar and a single basal appendage; in Chironomidae or Simuliidae larvae ...... 17 11( 10). Trichospores long-ellipsoidal with 2-4 appendages; zygospores <50 !lm long; thalli frequently attached to other gut organisms, especially to thalli of Paramoebidium ...... Simuliomyces microsporus 11'( 10). Trichospores ovoid to long-ovoid with 4-8 appendages; zygospores >50 !lm long; thalli always attached to hindgut cuticle ...... 12 12(11'). Base of mature thalli usually bifurcate, attached to hindgut cuticle by a mucilaginous secretion...... 13 12'(11 '). Base of mature thalli not bifurcate, attached to hindgut cuticle by a non-mucilaginous holdfast...... 14 13(12). Trichospores 39-72 x 8-10!lm; zygos pores 91-100 x 18-20 f.1m...... Pennella montana 13'(12). Trichospores 30-41 x 6.5-1O.5!lm;zygo spores 84-96 x 19-24 !lm...... Pennella simulii 14(12'). Trichospores 50-61 !lm long ...... Genistellospora guanacastensis 14'(12'). Trichospores < 50!lmlong ...... , .. 15 15(14'). Trichospores 38-41 !lmlong ...... Genistellospora nubila 15'(14'). Trichospores almost always <38 !lmlong (average length 27-30!lm) ...... 16 16(15'). Trichospores 24-41 !lmlong; zygospores 77-113 x 15-22 I-lm ...... Genistellospora homothallica 16'(15'). Trichospores 21-31 !lm long; zygospores 58-70 x 10-15!lm ...... : ...... Gen istellospora tepidaria 17(10'). Trichospores widest below midline (long-ovoid), 11-30 x 3-7 !lm, usually (but not always) in Culicidae larvae ...... Smittium culisetae 17'(10'). Trichospores longitudinally symmetrical (oval to subcylindrical), never in Culicidae larvae ...... 18 18(17'). Trichospores more or less cylindrical, 2-4 I-lmdiameter ...... 19 18'(17). Trichospores oval to elongate-oval, 3.5-10 I-lmdiameter ...... 21 19(18). In Simuliidae or Chironomidae larvae living in streams (lotic) ...... Smittium dipterorum 19'(18). In Chironomidae larvae living in phytotelms (usually bromeliads) or other lentic habitats ...... 20 20(19'). Thalli in fascicle-like clusters in hindgut, with some growth into midgut region...... Smittium fa sciculatum 20'(19'). Thalli not in fascicles and restrictedto hindgut region ...... Smittium phytotelmatum 21(18'). Base of thallus consisting of about 6 cells arranged in a ring...... Smittium annulatum 21 '(18'). Base of thallus without a ring of eells ...... 22 22(21 '). Triehospores about 3.5!lm diameter...... Smíttíum parvum 22'(21 '). Triehospores about 6-10 I-lmdiameter ...... Smittium culicisoides

DISCUSSION but disjunct life zones, altitudinal distributions, and temporal influenees that affeet the preva­ Though this report represents the results of lenee of aquatic insect hosts would have 110 colleetions in a variety of stream habitats required a differently designed and more over a period of years and at different seasons, lengthy study. Nonetheless, certain eonclu­ conclusions about distributional patterns of sions are justified from the data that have been Harpellales in Costa Rica are lirnitedby collee­ obtained. tion biases. Faetors sueh as presenee or First. it is quite clear that Harpellales infeet absenee of particular fungal speeies in similar many aquatic insect larvae throughout Costa Lichtwardt: Costa Rican gut fungi (Trichomycetes) 1379

Rica, but the degree of diversity and species found in several species of Baetis. Other richness of these fungi has not been thoroughly Baetidae were infected with what might be new explored. Harpella tica was found in over 95% genera of branched Harpellales, but they are of Simuliidae larvae and in a11 populations not described here because of insufficient doc­ sampled. This new species is probably not umentation. These were in nymphs of a Baetis endemic to Costa Rica, but the extent of its di s­ sp. from S fork R. Humo (Site 18) and a tribution in the Neotropics ís still to be deter­ Baetodes sp. from R. Macho (Site 10). In addi­ mined. Studies in southem Chile (Lichtwardt tion, what is possibly another new genus of and Arenas 1996) revealed a similar situatíon, Harpellales was found in an unidentified where another new species, H. meridianalis Caenidae nymph from Q. Sábalo (Site 3). Líchtw. & Arenas, was the only Harpella Twelve successful axenic cultures from encountered, but in high frequency. dipteran larvae represented seven new species Surprisingly, the very widespread species, H. of Smittium (three of them not named at this melusinae, which infects simuliid larvae in time). Smittium is the largest genus of many parts of the world, was found neither in Harpellales, currently containing 47 named Costa Rica nor in southem Chile. species plus the three unnamed ones described Other Harpellales infecting símuliid larvae and illustrated in this paper (Figs. 60-63). The were less distributed, and it is possible that eight cultured Costa Rican Smittiums can be some of the fungi that infect such larvae are distinguished from each other not only on a either restricted to particular stream conditions morphological basis, which was given primary or to particular species of hosts. Based on taxonomic emphasis, but also by their isozyme studies in other regions of the world, as well as pattems (Fig. 79), with the exception of S. culi­ Costa Rica, Trichomycetes in Simuliidae do cisoides isolate CR-253-12 and Smittium sp. #3 not in general appear to be species or even isolate CR-211-1. Those two had an 81% mea­ genus specific, but it was not possible in these sure of isozymic similarity, which normally investigations to separate habitat requirements would indicate species congruence, but they of particular species from host specificity. (It differ morphologically (compare Figs. 35-37 should be noted that the larval stages of many with Figs. 62, 63). Smittium culicisoídes, on aquatic insects cannot be identified to species the other hand, produces trichospores that are because of insufficient studies.) Among the essential1y indistinguishable from the common, thousands of simuliid larvae dissected, widespread S. culícis, but the isozyme analysis Genistellospora guanacastensis was found in Fig. 79 clearly indicates they aredist inct. only in four sites in Guanacaste Province, G. Water temperature is one of several factors nubila in one stream in Monteverde, and G. that influence a stream's fauna, and conse­ tepidaria in several streams at the La Selva quently the gut mycota. The warmest streams Biological Station. AIso apparentIy restricted at the various particular times collections were in distribution was the new monotypic genus made were on the Pacific side of the Divide: Graminelloides, found in two specíes of simuli­ Río Caracol, 27.5 C (Site 29), several streams id larvae from one stream near Portalón in southeast of Quepos, 26-27 C (Site 25), and R. Puntaren as Province. One expects that each of Piedras, 26-28 C (Site 30). At higher altitudes, these fungal species has a greater range than as might be expected, stream temperatures the collections indicate, but it is also evident were considerably cooler: Cordill era de that many gut fungi have not disseminated to Talamanca, 11-15 C (Sites 18·20); Monteverde the extent that H. tica has. Several other exam­ regíon, 15.5-17.5 C (Sites 6-9); premontane pIes of limited distributions are provided in the wet forest southwest of San José, 11-15 C preceding descriptions of individual fungal (Sites lS-17); and Wilson Botanical Garden, species. 19.5-20 e (Site 27). Streams in the low wet Trichomycetes in Costa Rican mayfly forest at La Selva (Sites 1-4) ranged from 24 to nymphs (Ephemeroptera) warrant additional 25.8 C. studies. In this paper the only Harpellales Another factor affecting stream biota is the reported from mayflies is the new · species content of organic matter. Our collections Spartiella anima. The relatively common íncluded pristine streams near the headwaters genus Paramoebidium (Amoebidiales) was of drainages with no nearby habitations, as well 1380 REVISTA DE BIOLOGlA TROPICAL

as streams obviously polluted by human activi­ of Ch ironomus larvae had consistently been ty. The most extreme case of pollution by found. What caused this phenomenon is not organic supplementation was found in Río known. On the next visit to that site several Guacimal in Monteverde downstream from years later, on 18 November 1991, both the where a cheese factory was piping whey into fauna and gut fungí were present in their usual the stream (Site 7). Small pieces of curd from high numbers. the whey effluent could be filtered in large Streams polluted with certain chemicals quantities from the malodorous water, and sub­ may, of course, have a profound negative effect merged leaves and rock surfaces were slimy. on the fauna and consequently on the presence Sorne of the particles were found in the guts of of trichomycete gut fungi. Such was the case Chironomidae larvae. The number of in a few streams visited by the author (not Chironomidae and Simuliidae Jarvae down­ reported in Table 1), most notably in the agri­ stream from the whey discharge was dramati­ cultural area within the general drainage of Río calIy greater than the number of these insects Estrella on the eastern coast north of Cahuita. immediately aboye the discharge, and the abun­ An almost complete lack of insect fauna, possi­ dance of Trichomycetes in their guts was also bly due to the widely reported use of agricul­ greater. In the stream below the effluentduri ng tural pesticides in that area, resulted in an the years of study the following Trichomycetes absence of trichomycete hosts. were found: Amoebidium colluviei, Hmpella Despite the abundance of larvae and gut tica, Genistellospora homothallica, fungí in the stretch of R. Guacimal cited aboye, Paramoebidium sp., Pennella montana, in most Costa Rican streams the overall density Símuliomyces microsporus, Smittium culi­ and species richness of aquatic insects with gut cisoídes, S. dipterorum, S. parvum, Stachylina fungi were not greater than that found by the grandíspora, and S. nana. In contrast, aboye author in many nontropical regions of the the effluent and at various sites along Q. world. What is often referred to as the latitudi­ Cuecha (the upper portion of R. Guacimal), nal species diversity gradient remains debat­ only five species were encountered: H. tica, G. able, both as to the causal factors and whether homothallíca, Paramoebídium sp., P. montana, the concept applies to all groups of organisms and, once, an undetermined species of (Illies 1969; Rosenzweig 1992). It has been Smittium. Obviously, the high organic content adequately demonstrated that many groups of in the water did not affect the growth and plants and animals have fewer species the far­ reproduction of many of the gut fungi. ther one gets from the Equator. Terrestrial The effect of this organic pollution on com­ insects are one such group, but is this true of position of the insect fauna was not easy to insects that have primaryaquatic stages? Sorne rneasure at the species level, because of the dif­ investigators have conc1uded that such a latitu­ ficulty in identifying larvae to species. Among dinal gradient possibly does exist among aquat­ the Chironomidae identified, one or more ic insects (Stout and Vandermeer 1975; species of the following genera were present: McElravy et al. 1981), others that a greater Chironomus, Cricotopus, Cryptochironomus, diversity of aquatic insects in the tropics is not Polypedilum, Cardiocladius, and an unidenti­ demonstrable (Patrick 1966; MacArthur 1972; fied genus of Orthocladiinae. Simuliidae Allan and Flecker 1993). In this discussion we included Simulium nr. callidium and several will focus on the species richness of unidentified or unidentifiable species. Sorne Harpellales in Costa Rica, which is, at the same time between 17 and 24 June 1988, however, time, a refIection of the diversity of their aquat­ the same stretch of R. Guacimal had undergone ic hosts, namely the larval stages of lower a dramatic change, involving a massive kill of Diptera (Nematocera), Ephemeroptera, and almost all insect larvae. This was indicated by Plecoptera. Excluded from the comments that the presence of many dozens of dead larvae in follow are terrestrial, marine, and freshwater sorne of the leaf packs (115 dead larvae con­ arthropods infected with Eccrinales and sisting of perhaps four species were counted in Asellariales, only a few of which were collect­ one leaf pack, for instance), and with almost no ed by the author because of the emphasis in this living larvae clinging to their usual substrates, study on Harpellales. nor within siltation zones where large numbers Seventeen species of Harpellales were Lichtwardt: Costa Rican gut fungi (Trichornycetlls) 1381

found in 36 Costa Rican streams during more the importance of sampling at least 20-25 rocks than 25 weeks of collecting from 1984 through (12-30 cm across in riffles of small streams) to 1991. In contrast, and as an example, collec­ obtain an adequate measure of species richness tions at five sites in two Rocky Mountain when comparing tropical and mid-Iatitude streams in Colorado, U.S.A. during a six-week streams. We used three of the same Costa period yielded 20 species of Harpellales Rican streams among those that were sampled (Lichtwardt and Williams 1988). If one con­ by Stout and Vandermeer: Quebrada Sábalo siders all species of Harpellales that have now (seven collections over 4 yr), Q. Esquina (six been found during two short investigations in coHections over 4 yr), and Río Corobicí (three that same region of the Rocky Mountains (a collections over 2 yr). Though the precise few species arestill to be described), they now locations of the stream riffles may have been total several dozen. Both the diversity of different, our rock samples yielded relatively stream life and species richness of particular few insect larvae, even when we once sampled groups of insects in the Rocky Mountains is 25 rocks, such that we did not try to tabulate well documented [Ward and Kondratieff 1992; the results. (Río Corobicí yielded almost no Peckarskyel al. (undated)]. insects in selected sites in 1988 and 1991, lead­ In our Costa Rican studies only nine genera ing to the suspicion that sorne detrimental event of Chironomidae larvae (species determina­ upstream had reduced the rich insect popula­ tions not possible) were found to be infected, tions reported by Stout and Vandermeer.) and consequently relatively few Costa Rican It would appear that the somewhat low Harpellales that inhabit such larvae have been number of Harpellales species found in Costa reported to date. With one exception, aH Rica, considering the temporal and spacial Ephemeroptera nymphs encountered in Costa extent of our investigations, might be attribut­ Rica were Baetidae, and only one fungal genus able to a relatively low diversity of the particu­ (Spartiella) is reported. In various nontropical lar kinds of aquatic insect taxa in which these parts of the world, Baetidae are known hosts of gut fungi have evolved. At the same time it seven of nine genera of Harpellales that infect should be noted that this study of tropical lotic ephemeropterans. The third order of harpellid Trichomycetes, because of the considerable hosts is Plecoptera, know to harbor six genera time it takes to dissect arthropods and prepare of Harpellales in nontropical regions. their gut fungi, does not represent all kinds of Plecopterans are species poor in Costa Rica, life zones-:-and certainly not all geographic because these insects are almost completely areas and microhabitats-that might have been restricted to cooler running waters (Illies 1969). explored in Costa Rica. The fact that more No Harpellales are reported from Costa Rica than two-thirds of the aquatic Trichomycetes plecopterans. that have been found to date in Costa Rica are A1though quantitative studies have not been new species, suggests that Costa Rica aud the carried out by the author, in his experience in Neotropics in general are reservoirs of many temperate North America, Australia, New yet unknown genera and species of these para­ Zealand, and Patagonia, kicking the substrate sites of arthropods. in suitable stream habitats and colIecting the released insects in a net usualIy has yielded, as one crude measure, a far greater variety and ACKNOWLEDGMENTS number of individual larvae that contain gut fungi than in most of the Costa Rican streams Support for this research is acknowledged that were sampled. (Of course, other more with appreciation from the National Science refined coliecting methods for particular kinds Foundation, Washington, DC, through grants of insect hosts of trichomycetes were also BSR-8019724, BSR-8516581, BSR-9006368, used.) Stout and Vandermeer (1975) sampled DEB-9220518, and DEB-952181L Laboratory streams in Costa Rica by colIecting individuals space and field facilities were provided by the from rocks in riffles and identifying the OTUs Organization for Tropical Studies at the La (operational taxonomic units) for nine orders of Selva Biological Station and the Las Cruces insects, and they also reported the average Biological Station, and by the Tropical Science number of individual s per rock. They stressed Center for research in the Monteverde Cloud 1382 REVISTADE BIOLOGIA TROPICAL

Forest Reserve. The University of Costa Rica Liehtwardt, R. W. 1994. Triehomycete fungi living in the faculty in the Escuela de Biología furnished guts of Costa Rican phytotelm larvae and other lentie dipterans. Rev. Biol. Trop. 42: 31-48. laboratory space for part of the research in 1984, and Elizabeth T. Lichtwardt assisted with Lichtwardt, R. W. 1996. Trichomyeetes and the arthropod the collection of insect specimens that year. gut, p. 315-330. In D. H. Howard and J. D. Miller The author is especially indebted for tíme-con­ (eds.). The Myeota. Human and animal relationships, suming identifications of insects by Leonard C. Vol. VI. Springer-Verlag, Berlin. Ferrington, Jr. (Chironornidae) and Paul Liechti Lichtwardt, R. W. & J. Arenas M. 1996. Triehomycetes in (Ephemeroptera) of the State Bíological Survey aquatic insects from southem Chile. Mycologia 88: of Kansas, and A.J. Shelley and staff of the 844-857. British Museum (Natural History) (Simuliidae). Lichtwardt, R. W., Y. Kobayasi & H. Indoh. 1987. Roger Grigg provided the isozyme data used to Trichomyeetesof Japan. Trans. Myeol. Soco Japan 28: generate the phenogram (Fig. 79); he also read 359-412. the manuscript and offered a number of sugges­ tions, as did Merlin White and J.K. Misra, for Lichtwardt, R. W. & M. C. Williams. 1988. Distribution which the author is grateful. Donald P. Rogers, and species diversity of trichomycete gut fungi in aquatic inseet larvae in two Rocky Mountain streams. Auburn, Washington, kindly translated the Can. 1. Bol. 66: 1259-1263. diagnoses of new taxa into Latin. Lichtwardt, R. W., and M. C. Williams. 1990. Trichomyeete gut fungi in Australian aquatie inseet larvae. Can. J. Bol. 68: \057-\074. REFERENCES López Lastra, C. C. 1990. Primera eita de Smittium mor­ Allan, J. D. & A. S. FleckeI. 1993. Biodiversity conserva­ bosum var. rioplatensis var. nov. (Trichomycetes: tion in running waters. BioScience 43: 32-43. Harpellales) patogeno de 5 especies de mosquitos (Diptera: Cu\icidae) en la Republica Argentina. Rev. Chatton, E. 1906. Sur la morphologie et l'évolution de Arg. Micologia 12: 14-18: I'Amoebidium repicola, nouvelle espece commensale de Daphnies. Arch. Zool. Exp. Gen., Ser. 4, 5:33-38. MacArthur, R. H. 1972. Geographical ecology: pattems in the distribution of species. Harper& Row, New York. Grigg, R. D. 1994. Isozyme variation in selected cultured 269 p. Trichomycetes. Ph.D. dissertation, University of Kansas. 182 pp. McE1ravy, E. P., V. H. Resh, H. Wolda & O. S. Flint, Jr. 1981. Diversity ofadult Triehoptera in a "non-season­ Grigg, R. & R. W. Lichtwardt. 1996. Isozyme pattems in al' tropical environment, p. 149-156. In G. P. Moretti cultured Harpellales. Mycologia 88: 219-229. (ed.). Ploceedings of the Third International Symposium on Trichoptera. DI. W. Junk Publishers, Hom, B. W. R. W. Lichtwardt. 1981. Studies on the & The Hague. nuttitional relationship of larval Aedes aegypti (Diptera: Culicidae) with Smittium culisetae Moss, S. T. & E. Descals. 1986. A previously undescribed (Trichomycetes). Mycologia 73: 724-940. stage in the \ife cycle of Harpellales (Trichomycetes). Mycologia 78: 213-222. Illies, 1. 1969. Biogeography and ecology of Neotropical freshwater insects, especially those from running Moss, S. T. & R. W. Lichtwardt. 1976. Development of waters, p. 685-708. In E. J. Fittkau, J. IIlies, H. trichospores and their appendages in Genistellospora Klinge, G. H. Schwabe, and H. Sioli (eds.). homothallica and other Harpellales and fine-structural Biogeography and ecology in South America. DI. W. evidence for the sporangial nature of trichospores. Junk Publishers, The Hague. Can. J. Bot 54: 2346-2364.

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